Combination therapies and methods of use thereof for treating cancer

ABSTRACT

Pharmaceutical compositions including an effective amount of an antiandrogen or androgen antagonist in combination with a Plk inhibitor and methods of use thereof for treating cancer are disclosed. Administration of the combination of the active agents can be effective to reduce cancer cell proliferation or viability in a subject with cancer to the same degree, or a greater degree than administering to the subject the same amount of either active agent alone. The active agents can be administered together or separately. Methods of selecting and treating subjects with cancers, particular prostate cancers including castration resistant prostate cancer, breast cancers, particularly androgen receptor positive breast cancers, and pancreatic cancers are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 61/932,283, filed onJan. 28, 2014, which is incorporated herein in its entirety.

FIELD OF THE INVENTION

The invention is generally directed to combination therapies includingan antiandrogen or androgen antagonist and a polo-like kinase inhibitorfor the treatment of cancer.

BACKGROUND OF THE INVENTION

Prostate cancer is the most frequently diagnosed non-skin related cancerand the second leading cause of cancer related deaths among men. Ahallmark of prostate cancer is its dependence on androgen signalingthrough the androgen receptor (AR). While the efficacy ofandrogen-depletion therapy for the treatment of metastatic prostatecancer has been known for more than 70 years, patients frequentlyprogress to androgen-independent or castrate-resistant prostate cancer(CRPC).

Several second line anti-androgen therapies have been developed whichfurther inhibit androgen signaling by competing with androgen for ARbinding, disrupting testosterone synthesis, or both. For example,abiraterone has been shown to bind to the androgen receptor (Richards etal., Cancer Res., 72:2176-2182 (2012). While beneficial, the response tothese strategies is almost always short lived.

There exists a need for improved therapies that effectively treatandrogen-independent or castrate-resistant prostate cancer, particularlythose cancers that are not effectively treated by the second-lineanti-androgen therapies discussed above.

There is also a need for therapies that effectively treat cancers whichoverexpress AR, or are otherwise dependent on the synthesis of steroidhormones for their growth and survival, such as some breast cancers.

Therefore, it is object of the invention to provide compositions andmethods of use thereof for treating androgen-dependent cancer that havebecome androgen-independent or castrate-resistant.

It is also an object of the invention to provide compositions andmethods of using thereof for treating cancers that overexpress AR orthat are otherwise dependent on steroid hormone synthesis.

SUMMARY OF THE INVENTION

Pharmaceutical compositions containing one or more antiandrogens orandrogen antagonists and one or more polo-like kinase (Plk) inhibitorsand methods of making and using thereof are described herein. Theantiandrogen(s) or androgen antagonist can be a steroid or asteroid-like molecule or a non-steroid. Anti-androgens or androgenantagonists, such as abiraterone, in combination with a polo-like kinase(Plk) inhibitor, such as BI2536, have shown a reduction in the viabilityand proliferation of cancer cells. The combination therapies can be usedto improve the initial efficacy of one or the other of the activeagents, or to re-sensitize cells that have become resistant to a dose(e.g., the maximum dose) of one or the other active agents when it isadministered alone. Specifically, the examples show that either agentadministered alone is ineffective at treating a cancer which has becomehormone-desensitized while the combination is extremely effective attreating such cancers.

Pharmaceutical compositions including an effective amount of acombination of an antiandrogen or androgen antagonist and a Plkinhibitor, or combinations thereof; and methods of use thereof fortreating cancer are disclosed. Typically, administration of thecombination of the two active agents (i.e., antiandrogen or androgenantagonist and Plk inhibitor) is effective to reduce cancer cellproliferation or viability in a subject with cancer to a greater degreethan administering to the subject the same amount of antiandrogen orandrogen antagonist alone or the same amount of Plk inhibitor alone. Inthe most preferred embodiments, the reduction in cancer cellproliferation or viability in the subject with cancer is more than theadditive reduction achieved by administering the antiandrogen orandrogen antagonist alone or the Plk inhibitor alone. In someembodiments, in subjects with tumors, the combination is effective toreduce tumor burden, reduce tumor progression, or a combination thereof.

In the preferred embodiment, the antiandrogen or androgen antagonist isabiraterone or a prodrug, analog, or derivative, or pharmaceuticallyacceptable salt thereof. In the most preferred embodiment, theabiraterone prodrug is abiraterone acetate. The dosage of abirateroneacetate can be, for example, 250-1,500 mg.

The Plk inhibitor is preferably a Plk1 inhibitor, for example, BI2536,Volasertib (BI 6727), GSK461364, HMN-176, HMN-214, rigosertib(ON-01910), MLN0905, or Ro3280. Preferred Plk1 inhibitors include BI2536and Volasertib. The dosage of BI25236 or Volasertib can be 1-500 mg,preferably at or below the maximum tolerated dose in a human.

These compositions and methods are particularly effective for treatingprostate cancer. In some embodiments, the prostate cancer isandrogen-dependent prostate cancer. In some embodiments, the prostatecancer is androgen-insensitive prostate cancer (e.g., castrate resistantprostate cancer). In some embodiments, the prostate cancer isinsensitive to hormone therapy, docetaxel, abiraterone when administeredalone, or one or more other first line or second line prostate cancertherapies.

These compositions and methods are also particularly effective fortreating breast cancer. In preferred embodiments the breast cancer is anandrogen receptor positive breast cancer. In some embodiments, thebreast cancer is estrogen and/or progesterone receptor positive. Inother embodiments the breast cancer is estrogen, progesterone, andandrogen-receptor negative. These compositions and methods are alsoeffective for treating non-hormonal cancers, such as pancreatic cancer,lung cancer and bowel cancer. In certain embodiments the cancers thatare sensitive to additive and more than additive effects of thecombination therapies are characterized by a specific gene profile. Forexample, cancer cells that express genes associated with the retinoicacid signaling receptor (RA) pathway can be more sensitive the effectsof the combination therapies than cancer cells that do not express thesegenes.

Methods of treating subjects in need there using these combinationtherapies are also provided. The antiandrogen or androgen antagonist andthe Plk inhibitor can be administered to the subject on the same day. Insome embodiments, the two agents are administered simultaneously. Theantiandrogen or androgen antagonist and the Plk inhibitor can be part ofthe same admixture, or administered as separate compositions. In someembodiments, the separate compositions are administered through the sameroute of administration. In other embodiments, the separate compositionsare administered through different routes of administration. Forexample, in some embodiments, an antiandrogen or androgen antagonist,such as abiraterone acetate, is administered orally, and a Plk1inhibitor is administered intravenously through injection or infusion.

In some embodiments, the antiandrogen or androgen antagonist isadministered to the subject prior to administration of the Plk inhibitorto the subject. The antiandrogen or androgen antagonist can beadministered to the subject, for example, 1, 2, 3, 4, 5, 6, 8, 10, 12,18, or 24 hours, 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, or 4 weeks, orany combination thereof prior to administration of the Plk inhibitor tothe subject.

In other embodiments, the Plk1 inhibitor is administered to the subjectprior to administration of the antiandrogen or androgen antagonist tothe subject. The Plk inhibitor can be administered to the subject, forexample, 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours, 1, 2, 3, 4, 5, 6,or 7 days, 1, 2, 3, or 4 weeks, or any combination thereof prior toadministration of the antiandrogen or androgen antagonist to thesubject.

In some embodiments, the combination therapy includes administering tothe subject one or more additional active agents. The active agent canbe a steroid, for example, prednisone which is often administered incombination with abiraterone. The second active agent can be achemotherapeutic agent, for example, docetaxel. In some embodiments, thedisclosed methods also include surgery or radiation therapy.

Methods for characterizing the gene expression profile of cancer cellsand/or the tumor microenvironment have also been developed to assess theextent to which the cancer cells or tumor associated cells are sensitiveto treatment with antiandrogens or androgen antagonists in combinationwith Plk inhibitors. These methods are useful in the diagnosis,prognosis, selection of patients, and the treatment of cancer. Forexample, patients having cancer cells that express components of theretinoic acid signaling pathway can be selected for treatment with thedisclosed therapies. In some embodiments, the combination therapyincludes methods for selecting patients who would be amenable forandrogen receptor and the Plk inhibitor combination therapies, and fortreating such patients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a model of androgen receptor signaling in prostate cancer. Theandrogen receptor (AR) is a nuclear receptor that responds to androgen(testosterone or dihydrotestosterone). Prostate cancer progresses from(1) prostate intraepithelial neoplasia, to (2) androgen-dependentprostate cancer, to (3) castrate-resistant prostate cancer (CRPC), tocancer resistant or insensitive to second line treatments such asabiraterone.

FIG. 2 is a diagram of the steps, intermediates and enzymes involved inthe steroid synthesis pathway and the mechanism by which abiraterone isbelieved to reduce or inhibit steroid synthesis. Specifically,abiraterone acetate inhibits CYP17A1 (as shown) and also acts as adirect androgen receptor antagonist (not illustrated).

FIG. 3A is a line graph showing the % viability of prostate cancer(LNCaP—androgen-sensitive human prostate adenocarcinoma cells) andcastrate resistant (CRPC) cells (C4-2) in the presence of increasingconcentrations of Plk1 inhibitor (BI2436 nM), and in the presence orabsence of synthetic androgen (R1881). FIG. 3B is a line graph showingthe proliferation (% relative to time 0) of prostate cancer cells(LNCaP—androgen-sensitive human prostate adenocarcinoma cells) and CRPC(C4-2) cells over time (hours) after administration of Plk1 inhibitor(BI2436 at 5 nM), and in the presence or absence of synthetic androgen(R1881).

FIG. 4 is a line graph showing proliferation (RFU following CelltiterGlo assay) of CRPC (C4-2) cells treated with DMSO, 5 nM BI2536, 10 μMabiraterone, or 5 nM BI2536 and 10 μM abiraterone over time (days postdrug administration).

FIGS. 5A, 5B, and SC are line graphs showing the % viability of prostatecancer cell lines LNCaP—androgen dependent cells (5A), 22RV1—castrateresistant (5B), and C4-2—castrate resistant (5C) in the presence ofincreasing concentrations of abiraterone (μM), and in the presence (10nm (

)) or absence (0 nm (

)) of Plk inhibitor (BI12536). The predicted additive result of the twoagents in combination is shown as “expected” (

).

FIGS. 6A and 6B are line graphs showing the % viability of breast cancercell lines BT20-AR− (6A) and AU565-AR+ (6B) in the presence ofincreasing concentrations of abiraterone (μM), and in the presence ((

) or absence (

) of Plk inhibitor (BI2536). The concentration of BI2536 used was 2 nMor 10 nM for AU565 and BT20, respectively. The predicted additive resultof the two agents in combination is shown as “expected” (

).

FIGS. 7A, 7B, and 7C are line graphs showing the % viability of prostatecancer cell lines LNCaP—androgen dependent cells in FBS (7A),22RV1—castrate resistant in csFBS (7B), and C4-2—castrate resistant incsFBS (7C) in the presence of increasing concentrations of abiraterone(μM), and in the presence (5 nm (

)) or absence (0 nm (

)) of docetaxel. The predicted additive result of the two agents incombination is shown as “expected” (

).

FIG. 8 is a bar graph showing the relative PSA mRNA expression ofprostate cancer cells 24 hours post-treatment with DMSO; 10 μMabiraterone; 5 nM Plk inhibitor BI2536; and 10 μM abiraterone/5 nM Plkinhibitor BI2536, respectively.

FIGS. 9A and 9B are bar graphs. FIG. 9A shows the average % mitoticcells of prostate cancer cell line LNCaP following exposure to DMSO; 10μM abiraterone; 2.5 nM Plk inhibitor BI2536; 2.5 nM Plk inhibitorBI2536/10 μM abiraterone; 1 μM docetaxel; and 1 μM docetaxel/10 μMabiraterone, respectively. FIG. 9B shows the relative change in PSAexpression during abiraterone treatment as a fold-difference over PSAexpression in DMSO at time points 0, 4, 8, 16, 24 and 48 hours,respectively.

FIGS. 10A-10I are line graphs demonstrating the % viability of SKBR3cells in the presence of increasing concentrations of abiraterone (μM),and in the presence of Plk inhibitor BI2536 at a range of concentrations(0.5 nm (FIG. 10A); 1 nm (FIG. 10B); 2 nm (FIG. 10C); 3 nm (FIG. 10D); 4nm (FIG. 10E); 5 nm (FIG. 10F); 7.5 nm (FIG. 10G); 10 nm (FIG. 10H); and10 nm (FIG. 10I), respectively (

)); or absence (0 nm (

)) of Plk inhibitor BI2536. The predicted additive result of the twoagents in combination is shown as “expected” (

).

FIG. 11 is a schematic heat map showing different breast cancer celllines (at top) in relation to expression of Her2; Androgen Receptor (AR;indicated by arrow at right of schematic); estrogen receptor alpha(ERα); and Progesterone Receptor (PGR). Breast cancer cell lines thatdisplayed more than additive efficacy of Abiraterone and Plk1 inhibitionare indicted by bold arrows.

FIG. 12 is a line graph, showing viability of LNCaP cells (

) and LNCaP-shMK2 cells (

) as a function of the % of untreated control in response to FBS; orvaried concentrations of the p38MAPK inhibitor SB203580 (SB; 5, 10, 20or 40 μM, respectively), in the presence or absence of 10 μM abiraterone(Abi).

FIGS. 13A and 13B are line graphs showing the effects of p38 inhibitionon Abiraterone sensitivity in prostate cancer cell lines. FIG. 13A showsCell viability (as % of untreated cells) of PCa cell lines (LNCap (

); 22Rv1 (

); and C4-2(

), respectively), in response to FBS and increasing doses (10, 20 and 40μM) of the p38MAPK inhibitor SB203580, respectively. FIG. 13B shows Cellviability (as % of untreated cells) of PCa cell lines (LNCap; 22Rv1; andC4-2, respectively), in response to increasing doses (10, 20 and 40 μM)of the p38MAPK inhibitor SB203580 (SB) with 10 μM abiraterone (Abi).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, the terms “combination therapy” refers to treatment of adisease or symptom thereof or a method for achieving a desiredphysiological change, including administering to an animal, such as amammal, especially a human being, an effective amount of two or morechemical agents or components to treat the disease or symptom thereof,or to produce the physiological change, wherein the chemical agents orcomponents are administered together, such as part of the samecomposition, or administered separately and independently at the sametime or at different times (i.e., administration of each agent orcomponent is separated by a finite period of time from each other).

As used herein, the term “dosage regime” refers to drug administrationregarding formulation, route of administration, drug dose, dosinginterval and treatment duration.

As used herein, the terms “individual”, “host”, “subject”, and “patient”are used interchangeably, and refer to a mammal, including, but notlimited to, primates, for example, human beings, as well as rodents,such as mice and rats, and other laboratory animals.

As used herein the term “effective amount” or “therapeutically effectiveamount” means a dosage sufficient to treat, inhibit, or alleviate one ormore symptoms of a disease state being treated or to otherwise provide adesired pharmacologic and/or physiologic effect. The precise dosage willvary according to a variety of factors such as subject-dependentvariables (e.g., age, immune system health, etc.), the disease, and thetreatment being administered. The effect of the effective amount can berelative to a control. Such controls are known in the art and discussedherein, and can be, for example the condition of the subject prior to orin the absence of administration of the drug, or drug combination, or inthe case of drug combinations, the effect of the combination can becompared to the effect of administration of only one of the drugs.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the therapeuticcompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

“Alkyl”, as used herein, refers to the radical of saturated orunsaturated aliphatic groups, including straight-chain alkyl, alkenyl,or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups,cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkylsubstituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, andcycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unlessotherwise indicated, a straight chain or branched chain alkyl has 30 orfewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chain,C₃-C₃₀ for branched chain), more preferably 20 or fewer carbon atoms,more preferably 12 or fewer carbon atoms, and most preferably 8 or fewercarbon atoms. Likewise, preferred cycloalkyls have from 3-10 carbonatoms in their ring structure, and more preferably have 5, 6 or 7carbons in the ring structure. The alkyl groups can also be substitutedwith one or more groups including, but not limited to, halogen, hydroxy,amino, thio, ether, ester, carboxy, oxo, and aldehyde groups. The alkylgroups may also contain one or more heteroatoms. “Lower alkyl”, as usedherein, means 1-6 carbons, preferably 1-5 carbons, more preferably 1-4carbons, most preferably 1-3 carbons.

The term “heteroalkyl”, as used herein, refers to straight or branchedchain, or cyclic carbon-containing radicals, or combinations thereof,containing at least one heteroatom. Suitable heteroatoms include, butare not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorousand sulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized. Heteroalkyls can be substituted as defined abovefor alkyl groups.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl, and—S-alkynyl. Representative alkylthio groups include methylthio,ethylthio, and the like. The term “alkylthio” also encompassescycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.“Arylthio” refers to aryl or heteroaryl groups.

“Alkenyl” and “Alkynyl”, as used herein, refer to unsaturated aliphaticgroups containing one or more double or triple bonds analogous in length(e.g., C₂-C₃₀) and possible substitution to the alkyl groups describedabove.

The terms “alkoxyl” or “alkoxy” as used herein refer to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, and —O-alkynyl. Aroxy can berepresented by —O-aryl or O-heteroaryl, wherein aryl and heteroaryl areas defined below. The alkoxy and phenoxyl groups can be substituted asdescribed above for alkyl.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein, R₉, R₁₀, and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈ or R₉ and R₁₀ taken together with the Natom to which they are attached complete a heterocycle having from 4 to8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In still more preferred embodiments, the term “amine” does notencompass amides, e.g., wherein one of R₉ and R₁₀ represents a carbonyl.In even more preferred embodiments, R₉ and R₁₀ (and optionally R′₁₀)each independently represent a hydrogen, an alkyl or cycloalkyl, analkenyl or cycloalkenyl, or alkynyl. Thus, the term “alkylamine” as usedherein means an amine group, as defined above, having a substituted (asdescribed above for alkyl) or unsubstituted alkyl attached thereto,i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “amide” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein, R₉ and R₁₀ are as defined above.

“Aryl” as used herein, refers to 5-, 6- and 7-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic (e.g.,biphenyl), or bihetereocyclic (e.g., bipyridinyl) ring system,optionally substituted with one or more substituents including, but notlimited to, by halogen, hydroxy, nitro, cyano, amino, primary,secondary, or tertiary amino, formyl, acyl, carboxylate, alkoxy,thioether, alkyl, alkenyl, and alkynyl, cycloalkyl, etc. Broadlydefined, “Ar”, as used herein, includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “Ar” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocycles. Examples of heterocyclic ring include, butare not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

“Aryl” includes substituted and unsubstituted biaryl and biheteroarylcompounds, optionally interrupted or bridged by one more atoms such ascarbon and/or heteroatoms (e.g., O, S, N, etc.). Examples include, butare not limited to, biaryl ethers, biaryl amines, biaryl thiols,biheteroaryl ethers, biheteroaryl amines and biheteroaryl thiols.

“Alkylaryl”, as used herein, refers to an alkyl group substituted withan aryl group (e.g., an aromatic or hetero aromatic group).

The term “carbocycle”, as used herein, refers to an aromatic ornonaromatic ring in which each atom of the ring is carbon.

“Heterocycle” or “heterocyclic”, as used herein, refers to a cyclicradical attached via a ring carbon or nitrogen of a monocyclic orbicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ringatoms, including carbon and one to four heteroatoms each selected fromthe group including non-peroxide oxygen, sulfur, and N(Y) wherein Y isabsent or is H, O, (C₁₋₄) alkyl, phenyl or benzyl, and optionallycontaining one or more double or triple bonds, and optionallysubstituted with one or more substituents. The term “heterocycle” alsoencompasses substituted and unsubstituted heteroaryl rings. Examples ofheterocyclic ring include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl,4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

“Heteroaryl”, as used herein, refers to a monocyclic aromatic ringcontaining five or six ring atoms including carbon and 1, 2, 3, or 4heteroatoms each selected from the group including non-peroxide oxygen,sulfur, and N(Y) where Y is absent or is H, O, (C₁-C₅) alkyl, phenyl orbenzyl. Non-limiting examples of heteroaryl groups include furyl,imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl,isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (orits N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl,isoquinolyl (or its N-oxide), quinolyl (or its N-oxide) and the like.The term “heteroaryl” can include radicals of an ortho-fused bicyclicheterocycle of about eight to ten ring atoms derived therefrom,particularly a benz-derivative or one derived by fusing a propylene,trimethylene, or tetramethylene diradical thereto. Examples ofheteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl,isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl,tetrazolyl, pyridyl (or its N-oxide), thientyl, pyrimidinyl (or itsN-oxide), indolyl, isoquinolyl (or its N-oxide), quinolyl (or itsN-oxide), and the like.

“Halogen”, as used herein, refers to fluorine, chlorine, bromine, oriodine.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein, X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, ancycloalkenyl, or an alkynyl, R′₁₁ represents a hydrogen, an alkyl, acycloalkyl, an alkenyl, an cycloalkenyl, or an alkynyl. Where X is anoxygen and R₁₁ or R′₁₁ is not hydrogen, the formula represents an“ester”. Where X is an oxygen and R₁₁ is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R₁₁ is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen and R′₁₁ is a hydrogen, the formula represents a “formate”. Ingeneral, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen; the formula represents a“thioester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R′₁₁ ishydrogen, the formula represents a “thioformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium. Other heteroatoms includesilicon and arsenic.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The term “substituted” as used herein, refers to all permissiblesubstituents of the compounds described herein. In the broadest sense,the permissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, halogens, hydroxyl groups, or any other organicgroupings containing any number of carbon atoms, preferably 1-14 carbonatoms, and optionally include one or more heteroatoms such as oxygen,sulfur, or nitrogen grouping in linear, branched, or cyclic structuralformats. Representative substituents include alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, andpolypeptide groups.

Heteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. It is understood that“substitution” or “substituted” includes the implicit proviso that suchsubstitution is in accordance with permitted valence of the substitutedatom and the substituent, and that the substitution results in a stablecompound, i.e. a compound that does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.

The term “pharmaceutically acceptable salt”, as used herein, refers toderivatives of the compounds defined herein, wherein the parent compoundis modified by making acid or base salts thereof. Example ofpharmaceutically acceptable salts include but are not limited to mineralor organic acid salts of basic residues such as amines; and alkali ororganic salts of acidic residues such as carboxylic acids. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. Suchconventional non-toxic salts include those derived from inorganic acidssuch as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, andnitric acids; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, naphthalenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, and isethionic salts.

The pharmaceutically acceptable salts of the compounds can besynthesized from the parent compound, which contains a basic or acidicmoiety, by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000, p. 704; and “Handbook of Pharmaceutical Salts:Properties, Selection, and Use,” P. Heinrich Stahl and Camille G.Wermuth, Eds., Wiley-VCH, Weinheim, 2002.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio.

“Prodrug”, as used herein, refers to a pharmacological substance (drug)which is administered in an inactive (or significantly less active)form. Once administered, the prodrug is metabolized in the body (invivo) into the active compound.

“Stereoisomer”, as used herein, refers to isomeric molecules that havethe same molecular formula and sequence of bonded atoms (constitution),but which differ in the three dimensional orientations of their atoms inspace. Examples of stereoisomers include enantiomers and diastereomers.As used herein, an enantiomer refers to one of the two mirror-imageforms of an optically active or chiral molecule. Diastereomers (ordiastereoisomers) are stereoisomers that are not enantiomers(non-superimposable mirror images of each other). Chiral moleculescontain a chiral center, also referred to as a stereocenter orstereogenic center, which is any point, though not necessarily an atom,in a molecule bearing groups such that an interchanging of any twogroups leads to a stereoisomer. In organic compounds, the chiral centeris typically a carbon, phosphorus or sulfur atom, though it is alsopossible for other atoms to be stereocenters in organic and inorganiccompounds. A molecule can have multiple stereocenters, giving it manystereoisomers. In compounds whose stereoisomerism is due to tetrahedralstereogenic centers (e.g., tetrahedral carbon), the total number ofhypothetically possible stereoisomers will not exceed 2n, where n is thenumber of tetrahedral stereocenters. Molecules with symmetry frequentlyhave fewer than the maximum possible number of stereoisomers. A 50:50mixture of enantiomers is referred to as a racemic mixture.Alternatively, a mixture of enantiomers can be enantiomerically enrichedso that one enantiomer is present in an amount greater than 50%.Enantiomers and/or diastereomers can be resolved or separated usingtechniques known in the art. “Chirality” also includes axial and planarchirality.

As used herein, the term “tumor cell” or “cancer cell”, denotes a cellwhich may be malignant (i.e., capable of metastasis and the mediation ofdisease), or benign. In contrast, a “non-tumor cell” is a normal cell(which may be quiescent or activated) that is located within a tumormicroenvironment, including but not limited to Tumor InfiltratingLymphocytes (TILs), leucocytes, macrophages, and/or other cells of theimmune system, and/or stromal cells, and/or fibroblasts (e.g., cancer ortumor associated fibroblasts).

The term “cell(s) of a tumor” is employed to refer to tumor cells andnon-tumor cells located within a tumor or a tumor environment. Thesubject (e.g., patient) and the tumors to be characterized in accordancewith the present disclosure may be of any mammalian species (e.g.,human, or primate, canine, feline, bovine, ovine, equine, porcine,rodent species (e.g., murine), etc.). The disclosure particularlyconcerns the characterization of human tumor cells as well as thecharacterization of human tumor microenvironments. The tumor cells ofrelevance to the present disclosure include, but are not limited to,tumor cells of cancers, including leukemias including, but not limitedto, acute leukemia, acute lymphocytic leukemia, acute myelocyticleukemias such as myeloblastic, promyelocytic, myelomonocytic,monocytic, erythroleukemia leukemias and myelodysplastic syndrome,chronic leukemias such as but not limited to, chronic myelocytic(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cellleukemia; polycythemia vera; lymphomas such as, but not limited to,Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as, butnot limited to, smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenström's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as, butnot limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing'ssarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma,periosteal sarcoma, soft-tissue sarcomas, angiosarcoma(hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma,liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovialsarcoma; brain tumors including but not limited to, glioma, astrocytoma,brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor,acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma,pineocytoma, pineoblastoma, primary brain lymphoma; breast cancerincluding, but not limited to, adenocarcinoma, lobular (small cell)carcinoma, intraductal carcinoma, medullary breast cancer, mucinousbreast cancer, tubular breast cancer, papillary breast cancer, Paget'sdisease, and inflammatory breast cancer; adrenal cancer, including butnot limited to, pheochromocytom and adrenocortical carcinoma; thyroidcancer such as but not limited to papillary or follicular thyroidcancer, medullary thyroid cancer and anaplastic thyroid cancer;pancreatic cancer, including but not limited to, insulinoma, gastrinoma,glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid orislet cell tumor, pituitary cancers including but not limited to,Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetesinsipius; eye cancers including, but not limited to, ocular melanomasuch as iris melanoma, choroidal melanoma, and cilliary body melanoma,and retinoblastoma; vaginal cancers, including, but not limited to,squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer,including but not limited to, squamous cell carcinoma, melanoma,adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease;cervical cancers including, but not limited to, squamous cell carcinoma,and adenocarcinoma; uterine cancers including, but not limited to,endometrial carcinoma and uterine sarcoma; ovarian cancers including,but not limited to, ovarian epithelial carcinoma, borderline tumor, germcell tumor, and stromal tumor, esophageal cancers including, but notlimited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma,plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma;stomach cancers including, but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; rectal cancers; liver cancers including, but not limited to,hepatocellular carcinoma and hepatoblastoma, gallbladder cancersincluding, but not limited to, adenocarcinoma; cholangiocarcinomasincluding, but not limited to, papillary, nodular, and diffuse; lungcancers including but not limited to, non-small cell lung cancer,squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer; testicular cancersincluding, but not limited to, germinal tumor, seminoma, anaplastic,classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancersincluding, but not limited to, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers including, but not limitedto, squamous cell carcinoma; basal cancers; salivary gland cancersincluding, but not limited to, adenocarcinoma, mucoepidermoid carcinoma,and adenoidcystic carcinoma; pharynx cancers including, but not limitedto, squamous cell cancer, and verrucous; skin cancers including, but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers including, but notlimited to, renal cell cancer, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor, bladder cancers including, but not limited to,transitional cell carcinoma, squamous cell cancer, adenocarcinoma,carcinosarcoma. In addition, cancers include myxosarcoma, osteogenicsarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma,synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,bronchogenic carcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, and gastriccancer (for a review of such disorders, see Fishman et al., 1985,Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al.,1997, Informed Decisions: The Complete Book of Cancer Diagnosis,Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc.,United States of America).

As used herein, the term “characterizing” is intended to refer toassessing a patient, tissue sample or cell for the expression of abiomarker and its presentation on the surface of or within a cell. Inaccordance with the principles of the present disclosure, suchcharacterization is mediated using molecules that physiospecificallybind, or that immunospecifically bind, to such expressed and presentedmolecules.

II. Compositions

The combination therapies include administration of an effective amountof at least two active agents, one being an antiandrogen or androgenantagonist and the other being a polo-like kinase inhibitor, to asubject in need thereof.

A. Active Agents

1. Antiandrogen or Androgen Antagonist

The combination therapies include an antiandrogen or androgenantagonist. The antiandrogen or androgen antagonist can reduce orinhibit the androgen synthesis pathway, or reduce or inhibit binding ofendogenous ligands including, but not limited to, testosterone anddihydrotestosterone (DHT), to the androgen receptor, or a combinationthereof. In preferred embodiments, the antiandrogen or androgenantagonist reduces or inhibits synthesis of testosterone or DHT. Forexample, the antiandrogen or androgen antagonist can reduce or inhibitthe pathway at one or more steps of the steroid synthetic pathwaysdepicted in FIG. 2. In some embodiments, the antiandrogen or androgenantagonist reduces or inhibits expression or activity of one or moreenzymes or cofactors in the pathway. In a preferred embodiment, theantiandrogen or androgen antagonist reduces expression or activity of 17alpha-hydroxylase, C17, 20-lyase, 5 alpha-reductase, or a combinationthereof.

In some embodiments, the antiandrogen or androgen antagonist reducesexpression or activity of the androgen receptor, reduces or inhibitsligand binding to the androgen receptor, reduces or inhibitstranslocation of the receptor to the nucleus, reduces or inhibits theactivity of the receptor in the nucleus, or a combination thereof. Forexample, the antiandrogen or androgen antagonist can target the androgenreceptor signaling pathway. In particular embodiments, the antiandrogenor androgen antagonist reduces or inhibits one or more of the signalingelements depicted in FIG. 1.

i. Steroidal Antiandrogen or Androgen Antagonists

In some embodiments, the antiandrogen or androgen antagonist is asteroid or has a steroidal structure. “Steroid” or “steroidal structure”as used herein typically refers to molecules having the ABCD ringcharacteristic of steroids.

In one embodiment, the antagonist has the formula:

Where X represent the A, B, and C rings of a steroid, R₁₄ is hydrogen,R₁₅ is hydrogen or C₁-C₆, preferably C₁-C₄ substituted or unsubstitutedalkyl or alkoxy, hydroxy, or alkylcarbonyloxy having 2-6, preferably 2-5carbons or R₁₄ and R₁₅ together represent a double bond, R₁₆ is hydrogenor C₁-C₆, preferably C₁-C₄ substituted or unsubstituted alkyl, and Y isa substituted or unsubstituted heterocycle or fused heterocycle. In someembodiments, Y is a substituted or unsubstituted fused heterocycle.

In a particular embodiment, the antagonist is TOK-001 which has thefollowing structure:

In one embodiment, the antagonist has the formula:

Where X represent the A, B, and C rings of a steroid, R is a hydrogen orC₁-C₆, preferably C₁-C₄ substituted or unsubstituted alkyl, R₁₄ ishydrogen, R₁₅ is hydrogen or C₁-C₆, preferably C₁-C₄ substituted orunsubstituted alkyl or alkoxy, hydroxy, or alkylcarbonyloxy having 2-6,preferably 2-5 carbons or R₁₄ and R₁₅ together represent a double bond,and R₁₆ is hydrogen or C₁-C₆, preferably C₁-C₄ substituted orunsubstituted alkyl.

In the most preferred embodiments, the antiandrogen or androgenantagonist is abiraterone, or a prodrug, analog, or derivative, orpharmaceutically acceptable salt thereof. The structure of abirateroneis shown below:

Abiraterone, as well as prodrugs, analogs, derivatives, orpharmaceutically acceptable salts thereof are known in the art. See, forexample, U.S. Pat. No. 5,604,213 which is specifically incorporated byreference herein in its entirety.

Abiraterone inhibits 17α-hydroxylase/C17,20 lyase (CYP17A1), an enzymewhich is expressed in testicular, adrenal, and prostatic tumor tissues.CYP17 catalyzes two sequential reactions: (a) the conversion ofpregnenolone and progesterone to their 17-α-hydroxy derivatives by its17α-hydroxylase activity, and (b) the subsequent formation ofdehydroepiandrosterone (DHEA) and androstenedione, respectively, by itsC17,20 lyase activity (see FIG. 2). DHEA and androstenedione areandrogens and precursors of testosterone, therefore, inhibition of CYP17activity by abiraterone decreases circulating levels of testosterone. Itis also believed that abiraterone acts as a direct androgen receptorantagonist.

Suitable dosages of abiraterone when used as a first or second linetherapy for treatment of cancer are also known in the art. For example,U.S. Pat. No. 5,604,213 teaches that a therapeutically effective dosecan be in the range 0.001-0.04 mmole/kg body weight, preferably0.001-0.01 mmole/kg, administered daily or twice daily during the courseof treatment. In some embodiments the dosage is 10-2,000 mg/patient perday, 100-1,500 mg/patient per day, 250-1,250 mg/patient per day, or500-1000 mg/patient per day.

In a particular embodiment, the abiraterone is formulated as a prodrugsuch as abiraterone acetate. Following administration, abirateroneacetate is converted into the active form, abiraterone. It is believedthis conversion is esterase-mediated and not dependent on CYP.Abiraterone acetate is a lipophilic compound with an octanol-waterpartition coefficient of 5.12 (Log P) and is practically insoluble inwater. Abiraterone acetate is sold under the trade name ZYTIGA®. Atablet for oral administration includes 250 mg abiraterone acetate andinactive ingredients including colloidal silicon dioxide, croscarmellosesodium, lactose monohydrate, magnesium stearate, microcrystallinecellulose, povidone, and sodium lauryl sulfate. The recommend dailydosage is 1000 mg per day (e.g., four tablets of ZYTIGA®), but can beincreased or decreased depending on the condition of the subject to betreated. For example, the dosage or dosing frequency is often decreased(e.g., to 250 mg/day, 500 mg/day, 750 mg/day, etc.,) if the subject isexperiencing hepatotoxicity, or when the drug is coadministered with aCYP2D6 substrate. Alternatively, the dosage or dosing frequency can beincreased when administered in combination with a strong CYP3A4 inducer.

For the treatment of prostate cancer, abiraterone is often administeredin combination with a steroid such as prednisone or prednisolone. Thesteroid is given to reduce the chances of (1) fluid retention, (2)raised blood pressure, or (3) decreased levels of potassium in the bloodas a result of the abiraterone treatment. Suitable prednisonecompositions and dosages for use in combination with abiraterone areknown in the art. For example, a recommended prednisone co-therapy is 5mg administered orally twice daily.

ii. Non-Steroidal Antidrogens and Androgen Antagonists

In some embodiments, the antiandrogen or androgen antagonist is not asteroid or does not have a steroidal structure. In one embodiment, thecompound has the structure:

wherein,

ring A is monocyclic heteroaryl, bicyclic heteroaryl, or naphthyl;

m is 0, 1, 2, 3 or 4;

each R^(A) is independently selected from H, halogen, —CN, —NO₂, —OH,—OR⁹, —SR⁹, —S(═O)R¹⁰, —S(═O)₂R¹¹, —N(R¹¹)S(═O)₂R¹⁰, —S(═O)₂N(R⁹)₂,—C(═O)R¹⁰, —OC(═O)R¹⁰, —CO₂R⁹, —N(R⁹)₂, —C(═O)N(R⁹)₂, substituted orunsubstituted C₁-C₆alkyl, substituted or unsubstituted C₁-C₆fluoroalkyl, substituted or unsubstituted C₁-C₆ fluoroalkoxy,substituted or unsubstituted C₁-C₆ alkoxy, substituted or unsubstitutedC₁-C₆ heteroalkyl, substituted or unsubstituted C₃-C₁₀ cycloalkyl,substituted or unsubstituted C₂-C₁₀ heterocycloalkyl, substituted orunsubstituted phenyl or substituted or unsubstituted monocyclicheteroaryl;

each R¹ is independently selected from H, —OH, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C1-C₆ alkoxy,and substituted or unsubstituted C₁-C₆ fluoroalkyl;

or both R¹ are taken together with the carbon atom to which they areattached to form a substituted or unsubstituted C₃-C₁₀ cycloalkyl or asubstituted or unsubstituted C₂-C₁₀ heterocycloalkyl;

each R² is H; or both R² are taken together with the carbon to whichthey are attached to form —C(═S)— or —C(═O)—;

each R³ is H; or both R³ are taken together with the carbon to whichthey are attached to form —C(═S)— or —C(═O)—; provided that each R² isnot H if each R³ is H;

ring B is phenyl, naphthyl, monocyclic heteroaryl, or bicyclicheteroaryl;

n is 0, 1, 2, 3 or 4;

each R⁴ is independently selected from H, halogen, —CN, —NO₂, —OH, —OR⁹,—SR⁹, —S(═O)R¹⁰, —S(═O)₂R¹⁰, —N(R₁₁)S(═O)₂R¹⁰, —S(═O)₂N(R⁹)₂, —C(═O)R¹⁰,—OC(═O)R¹⁰, —CO₂R⁹, —OCO₂R¹⁰, —N(R⁹)₂, —C(═O)N(R⁹)₂, —OC(═O)N(R⁹)₂,—NR¹¹C(═O)N(R⁹)₂, —NR¹¹C(═O)R¹⁰, —NR¹¹C(═O)OR¹⁰, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₁-C₆fluoroalkyl, substituted or unsubstituted C₁-C₆ fluoroalkoxy,substituted or unsubstituted C₁-C₆ alkoxy, and substituted orunsubstituted C₁-C₆ heteroalkyl;

R⁵ is substituted or unsubstituted C₂-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ fluoroalkyl, substituted or unsubstituted C₂-C₁₀alkoxy, substituted or unsubstituted C₂-C₁₀ fluoroalkoxy, substituted orunsubstituted C₂-C₁₀ heteroalkyl, substituted or unsubstituted C₂-C₁₀heterofluoroalkyl, or -L¹-L²-R⁶;

L¹ is absent, —O—, —S—, —S(═O)—, —S(═O)₂—, —NH—, —C(═O)—, —C(═O)NH—,—NHC(═O)—, —NHC(═O)O—, —NHC(═O)NH—, —C(═O)O—, —OC(═O)—, —OC(═O)O—,—OC(═O)NH—, —NHS(═O)₂—, or —S(═O)₂NH—;

L² is substituted or unsubstituted C₁-C₆ alkylene, substituted orunsubstituted C₁-C₆ fluoroalkylene or substituted or unsubstituted C₁-C₆heteroalkylene;

R⁶ is —CN, —NO₂, —OH, —OR⁹, —SR⁹, —S(═O)R¹⁰, —S(O)₂R¹⁰,—N(R₁₁)S(═O)₂R¹⁰, —S(═O)₂N(R⁹)₂, —C(═O)R¹⁰, —OC(═O)R¹⁰, —CO₂R⁹,—OCO₂R¹⁰, —N(R⁹)₂, —C(═O)N(R⁹)₂, —OC(═O)N(R⁹)₂, —NR¹¹C(═O)N(R⁹)₂,—NR¹¹C(═O)R¹⁰, —NR¹¹C(═O)OR¹⁰, substituted or unsubstitutedC.sub.1-C.sub.6alkyl, substituted or unsubstituted C₁-C₆ fluoroalkyl,substituted or unsubstituted C₁-C₆ heteroalkyl, substituted orunsubstituted C₃-C₁₀ cycloalkyl, substituted or unsubstituted C₂-C₁₀heterocycloalkyl, substituted or unsubstituted monocyclic heteroaryl,substituted or unsubstituted bicyclic heteroaryl, substituted orunsubstituted phenyl, or substituted or unsubstituted naphthyl;

each R⁹ is independently selected from H, substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted C₁-C₆ heteroalkyl, substitutedor unsubstituted C₁-C₆ fluoroalkyl, substituted or unsubstituted C₃-C₁₀cycloalkyl, substituted or unsubstituted C₂-C₁₀ heterocycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, C₁-C₄ alkylene-(substituted or unsubstituted C₃-C₁₀cycloalkyl), —C₁-C₄ alkylene-(substituted or unsubstituted C₃-C₁₀heterocycloalkyl), —C₁-C₄ alkylene-(substituted or unsubstituted aryl),and —C₁-C₄ alkylene-(substituted or unsubstituted heteroaryl); or

two R⁹ groups attached to the same N atom are taken together with the Natom to which they are attached to form a substituted or unsubstitutedC₂-C₁₀ heterocycloalkyl;

R¹⁰ is substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted C₁-C₆ heteroalkyl, substituted or unsubstituted C₁-C₆fluoroalkyl, a substituted or unsubstituted C₃-C₁₀ cycloalkyl, asubstituted or unsubstituted C₂-C₁₀ heterocycloalkyl, a substituted orunsubstituted aryl, a substituted or unsubstituted benzyl, a substitutedor unsubstituted heteroaryl, —C₁-C₄ alkylene-(substituted orunsubstituted C₃-C₁₀ cycloalkyl), —C₁-C₄ alkylene-(substituted orunsubstituted C₂-C₁₀ heterocycloalkyl), —C₁-C₄ alkylene-(substituted orunsubstituted aryl), or —C₁-C₄ alkylene-(substituted or unsubstitutedheteroaryl);

R¹¹ is H or C₁-C₄ alkyl.

These compounds are described in U.S. Patent Application Publication No.2013/0116258, which is incorporated herein by reference in its entirety.

In some embodiments, the compound is ARN-509 which has the followingstructure:

2. Polo-Like Kinase Inhibitor

The combination therapies include one or more polo-like kinase (Plk)inhibitors. Polo-like kinases (Plks) are a family of conservedserine/threonine kinases involved in the regulation of cell cycleprogression through G2 and mitosis. The catalytic domain of polo-likekinases is located in the N-terminus. The C-terminus of Plks containsone or two motifs known as polo boxes that help localize the kinase tospecific mitotic structures during mitosis. These include thecentrosomes in early M phase, the spindle midzone in early and lateanaphase and the midbody during cytokinesis.

Mammalian polo-like kinases include Plk1, Plk2/Snk, Plk3/Prk/FnK,Plk4/Sak, and Plk5. The polo-like kinase inhibitor can reduce or inhibitexpression or activity of Plk1, Plk2/Snk, Plk3/Prk/FnK, Plk4/Sak, andPlk5. For example, in some embodiments the inhibitor reduces or inhibitsPlk1, Plk2/Snk, Plk3/Prk/FnK, Plk4/Sak, and/or Plk5 mRNA or proteinexpression. In some embodiments, the polo-like kinase inhibitor reducesor inhibits the kinase activity of Plk1, Plk2/Snk, Plk3/Prk/FnK,Plk4/Sak, and/or Plk5. In some embodiments, the polo-like kinaseinhibitor reduces or inhibits expression of more than one polo-likekinase, for example by targeting a conserves region of the proteins suchas the polo box(es).

a. Small Molecule PLK Inhibitors

In a preferred embodiment, the polo-like kinase inhibitor (Plkinhibitor) is a small molecule. “Small molecule” as used herein, refersto an organic molecule, inorganic molecule, or organometallic moleculehaving a molecular weight less than 2000, 1500, 1200, 1000, 750, or 500atomic mass units. Polo-like kinase inhibitors are known in the art andinclude, for example, BI2536, Volasertib (BI 6727), GSK461364, HMN-176,HMN-214, rigosertib (ON-01910), MLN0905, and Ro3280, several of whichare discussed in Medema, et al., Clin. Cancer Res., 17:6459-6466 (2011),which is specifically incorporated by reference herein in its entirety.

Each of the Plk inhibitors, preferred dosages and routes ofadministration are discussed in more detail below, however, generally,the compounds can be administered to humans in an amount from about0.0001 mg/kg of body weight to about 100 mg/kg of body weight per day.Generally, for intravenous injection or infusion, dosage may be lowerthan for other methods of delivery.

Some of the Plk inhibitors have been investigated for anti-cancerproperties in preclinical experiments and clinical trials. In someembodiments, the dosage of Plk inhibitor used in combination therapiesis the same as a dosage used to treat or prevent a cancer in a clinicaltrial, or a human equivalent to a dosage used to treat cancer in ananimal study. However, the Examples below illustrate that the more thanadditive effect of the combination therapy is not the result of G2/Marrest induced by the Plk inhibitor. Therefore, in some embodiments, thedosage is different than the dosage used to treat cancer. For example,the dosage can be lower than the dosage used to treat cancer, or thedosage can be higher than the dosage used to treat cancer provided thatthe dosage is safe and tolerable to the subject. Preferably, the dosageis at or below a maximum tolerated dose as determined in a clinicaltrial. In some embodiments, the maximum tolerated dose is 250 mg.

i. Dihydropteridinones

In some embodiments, the Plk inhibitor has the formula described in U.S.Pat. No. 6,806,272, which is incorporated herein reference in itsentirety. The compounds have the formula:

wherein

R¹ denotes a group selected from among hydrogen, NH₂, XH, halogen and aC₁-C₃-alkyl group optionally substituted by one or more halogen atoms,

R₂ denotes a group selected from among hydrogen, CHO, XH, —X—C₁-C₂-alkyland an optionally substituted C₁-C₃-alkyl group,

R³, R⁴ which may be identical or different denote a group selected fromamong optionally substituted C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl,C₂-C₁₀-alkynyl, aryl, heteroaryl, C₃-C₈-cycloalkyl,C₃-C₈-heterocycloalkyl, —X-aryl, —X-heteroaryl, —X-cycloalkyl,—X-heterocycloalkyl, —NR⁸-aryl, —NR⁸-heteroaryl, —NR⁸-cycloalkyl and—NR⁸-heterocycloalkyl, or a group selected from among hydrogen, halogen,COXR⁸, CON(R⁸)₂, COR⁸ and XR⁸, or R³ and R⁴ together denote a 2- to5-membered alkyl bridge which may contain 1 to 2 heteroatoms,

R⁵ denotes hydrogen or a group selected from among optionallysubstituted C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, aryl,heteroaryl and —C₃-C₆-cycloalkyl, or R³ and R⁵ or R⁴ and R⁵ togetherdenote a saturated or unsaturated C₃-C₄-alkyl bridge which may contain 1to 2 heteroatoms, R⁶ denotes optionally substituted aryl or heteroaryl,R⁷ denotes hydrogen or —CO—X—C₁-C₄-alkyl, and X in each caseindependently of one another denotes O or S, R⁸ in each caseindependently of one another denotes hydrogen or a group selected fromamong optionally substituted C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄ alkynyland phenyl,

optionally in the form of the tautomers, the racemates, the enantiomers,the diastereomers and the mixtures thereof, and optionally thepharmacologically acceptable acid addition salts thereof.

Specific compounds of the formula above and other Plk inhibitors aredescribed below.

BI2536

In a preferred embodiment, the Plk inhibitor is BI2536, or a prodrug,analog, or derivative, or pharmaceutically acceptable salt thereof.BI2536 has the structure:

BI2536 is a potent Plk1 inhibitor with IC50 of 0.83 nM (Steegmaier, etal., Current Biology, 17:316-322 (2007)). It shows 4- and 11-foldgreater selectivity against Plk2 and Plk3. In preclinical experimental,BI2536 was given i.v. once or twice per week was highly efficacious indiverse xenograft models with acceptable tolerability. The drug wasbelieved to work by inhibiting cell proliferation through a mitoticarrest, and subsequently induction of tumor-cell death. Administrationof BI2536 at 50 mg/kg once or twice per week significantly inhibitedgrowth of HCT 116 xenografts with T/C of 15% and 0.3%, respectively.BI2536 treatment twice-weekly also lead to excellent tumor-growth inBxPC-3 and A549 models with T/C of 5% and 14%, respectively((Steegmaier, et al., Current Biology, 17:316-322 (2007)).

BI2536 has been the subject of a number of clinical travels testing thesafety and efficacy of the drug in a range of dosages and regimes andfor treatment of a number of cancers. For example, in a randomized,open-label, phase I/II trial to investigate the maximum tolerated doseof the Polo-like kinase inhibitor BI2536 in elderly patients withrefractory/relapsed acute myeloid leukemia, 68 elderly patients withrelapsed/refractory AML were administered BI2536 on one of threeschedules (day 1, days 1-3, and days 1+8). The maximum tolerated dosewas 350 mg and 200 mg in the day 1 and days 1+8 schedules, respectively.The day 1-3 schedule appeared equivalent to the day 1 schedule and wasdiscontinued early (Muller-Tidow, et al., Br. J. Haematol.,163(2):214-22 (2013)). Likewise, a phase I open-label dose-escalationstudy tested the maximum tolerated dose of intravenous BI2536 togetherwith pemetrexed in previously treated patients with non-small-cell lungcancer. The patients received 500 mg/m² pemetrexed and escalating dosesof BI2536 on day 1 every 3 weeks. Forty-one patients received BI2536(100-325 mg). Two dose-limiting toxicities (DLT) occurred at BI2536 325mg (grade 3 pruritus and rash; grade 4 neutropenia). Therefore, themaximum tolerated dose (MTD) for BI2536 in combination with pemetrexedwas 300 mg (Ellis, et al., Clin. Lung Cancer, 14(1):19-27 (2013) Epub2012 Jun. 1). BI2536 at 200 mg combined with standard-dose pemetrexedwas determined to have an acceptable safety profile. Other studies havesuggested a lower MTD, e.g., 50-70 mg (Frost, et al., Curr. Oncology,19(1):e25-35 (2012)).

An open, randomized, clinical phase II trial in patients withun-resectable advanced pancreatic cancer investigating the efficacy,safety, and pharmacokinetics of BI 2536 administered in repeated 3-weekcycles as a single i.v. dose of 200 mg on day 1 or as 60 mg doses ondays 1, 2, and 3 is currently underway.

Therefore, in some embodiments, BI2536 is administered to a subject 1,2, 3, or more times a week in a dosage of about 1-500 mg, preferablyabout 10-400 mg, more preferably about 50-350 mg, most preferably 60-300mg. In a particular embodiment the dosage is 50, 100, 150, 200, 250,300, or 350 mg of BI2536 administered to a subject once, twice, threetimes or more than three times a week, or once every two, three or fourweeks. In some embodiments, BI2536 is administered by intravenousinjection or infusion.

Volasertib (BI6727)

Like BI2536, BI6727 is an ATP-competitive kinase inhibitor from thedihydropteridinone class of compounds. BI6727 is a highly potent Plk1inhibitor with IC50 of 0.87 nM. It also shows 6- and 65-fold greaterselectivity against Plk2 and Plk3. BI6727 at concentrations up to 10 μMdisplays no inhibitory activity against a panel of >50 other kinases invitro (Rudolph D, et al. Clin. Cancer Res., 15(9), 3094-3102 (2009)).BI6727 has the structure:

Preclinical experiments in a mouse model show that administration ofBI6727 at ˜25 mg/kg/day significantly inhibits the growth of multiplehuman carcinoma xenografts including HCT116, NCI-H460, andtaxane-resistant CXB1 colon carcinoma, accompanied by an increase in themitotic index as well as an increase in apoptosis (Rudolph D, et al.Clin. Cancer Res., 15(9), 3094-3102 (2009)). Some in vivo studiesindicate that BI6727 exhibits a better toxicity and pharmacokineticprofile than BI12536 (Harris, et al., BMC Cancer, 12, 80 (2012)).

BI6727 has been the subject of a number of clinical travels testing thesafety and efficacy of the drug in a range of dosages and regimes andfor treatment of a number of cancers. A phase I first-in-humans study ofvolasertib was conducted in 65 patients with advanced solid tumors,including 10 with NSCLC. Volasertib was administered i.v. once every 3weeks following a dose-escalation design (12-450 mg). The study reportedneutropenia, thrombocytopenia, and febrile neutropenia as DLTs and anMTD of 400 mg (Gil, et al., J. Clin. Oncol., 28 Suppl 15:abstr 3061(2010), Schoffski, et al., Eur. J. Cancer, 48(2):179-86 (2012)). 300 mgwas the recommended dose for further development based on overalltolerability. In a phase I study of volasertib (BI 6727) combined withafatinib (BIBW 2992) in advanced solid tumors, the MTD was determined tobe 300 mg of BI 6727, when administered in combination with afatinib(Peeters, et al., J. Clin. Oncol, 31 (suppl; abstr 2521) (2013)).

Therefore, in some embodiments, BI 6727 is administered to a subject 1,2, 3, or more times a week in a dosage of between about 1-600 mg,preferably about 10-500 mg, more preferably about 50-400 mg, mostpreferably 100-350 mg. In a particular embodiment the dosage is 50, 100,150, 200, 250, 300, 350, or 400 mg of BI 6727 administered to a subjectonce, twice, three times or more than three times a week, or once everytwo, three or four weeks. In some embodiments, BI 6727 is administeredby intravenous injection or infusion.

ii. Other Classes of Plk Inhibitors

The inhibitor may be a molecule other than a dihydropteridinones. Otherclasses of inhibitors include, but are not limited to, pyridopyrimidines(see U.S. Patent Application Publication No. 2010/004141 and WO2009/112524), aminopyrimidines (see U.S. Patent Application PublicationNo. 2010/010014), substituted thiazolidinones (see European PatentApplication No. EP 2141163), pteridine derivatives (see European PatentApplication No. EP 2079743), dihydroimidazo[1,5-f]pteridines (see WO2010/025073), metasubstituted thiazolidinones, (see U.S. PatentApplication Publication No. 2010/048891), benzyl styryl sulfoneanalogues (see WO 2009/128805), and stilbene derivatives.

The applications cited above are incorporated herein by reference intheir entirety.

Specific inhibitors are discussed below:

GSK461364

GSK461364 inhibits purified Plk1 with Ki of 2.2 nM. It is more than1000-fold selective against Plk2/3.

The structure for GSK461364 is

Cell culture growth inhibition by GSK461364 can be cytostatic orcytotoxic but leads to tumor regression in xenograft tumor models underproper dose scheduling. In an animal model, dosages of 25, 50, and 100mg/kg were administered via i.p. every 2 days or every 4 days(Gilmartin, et al., Cancer Res, 69(17), 6969-6977 (2009)).

A phase I first-in-humans study of GSK461364 was conducted in 27patients with advanced solid tumors (Olmos, et al., Clin. Cancer Res.,17:3420-30 (2011)). The agent was administered i.v. following 2schedules with different dosing (50-225 mg on days 1, 8, and 15(schedule A) or 25-100 mg on days 1, 2, 8, 9, 15, and 16 (schedule B) ona 28-day cycle. DLTs included grade 4 neutropenia, sepsis, and pulmonaryembolism. The final recommended phase 1 dose for GSK461364 was 225 mgadministered intravenously in schedule A. Because of the high incidence(20%) of venous thrombotic emboli (VTE), coadministration ofprophylactic anticoagulation agent is recommended.

Therefore, in some embodiments, GSK461364 is administered to a subject1, 2, 3, or more times a week in a dosage of between about 1-400 mg,preferably about 10-350 mg, more preferably about 25-300 mg, mostpreferably 25-225 mg. In a particular embodiment the dosage is 50, 100,150, 200, 250, 300, 350, or 400 mg of GSK461364 administered to asubject once, twice, three times or more than three times a week, oronce every two, three or four weeks. In some embodiments, GSK461364 isadministered by intravenous injection or infusion.

HMN-176 and HMN-214

HMN-176 is a stilbene derivative that is an active metabolite of theprodrug HMN-214. It does not directly inhibit the enzymatic activity ofPlk1 but rather affects subcellular distribution of Plk1. The structuresof HMN-176 and HMN-214 are (A) and (B) respectively:

HMN-176 shows potent cytotoxicity toward various human tumor cell lines,and in mitotic cells, it causes cell cycle arrest at M phase through thedestruction of spindle polar bodies, followed by the induction of DNAfragmentation. In preclinical experiments it was a potent antitumoractivity in mouse xenograft models when administered at dosage of 10mg/kg and 20 mg/kg on days 1 and 28 (Tanaka, et al., Cancer Res.,63:6942-6947 (2003).

A phase I pharmacokinetic study of HMN-214 in patients with advancedsolid tumors, thirty-three patients were enrolled onto four dosingcohorts of HMN-214 from 3 to 9.9 mg/m²/d using a continuous 21-daydosing schedule every 28 days. A severe myalgia/bone pain syndrome andhyperglycemia were dose-limiting toxicities at 9.9 mg/m²/d, and themaximum tolerated dose and recommended dose on this schedule wasdetermined to be 8.0 mg/m²/d (Garland, et al., Clin. Cancer Res., 1;12(17):5182-9 (2006)).

In another study, DLTs of prolonged neutropenia, febrile neutropenia,neutropenic sepsis, electrolyte disturbance, neuropathy, and myalgiawere observed at doses of 24 to 48 mg/m² for 5 consecutive days every 4weeks. MTD was established at the range of 18 to 30 mg/m², based onprevious patient treatment load (Patnaik, J. Clin. Oncol, 22 Suppl:abstr514.).

Therefore, in some embodiments, HMN-214 (or HMN-176) is administered toa subject 1, 2, 3, 4, 5, 6, or 7 times a week in a dosage of betweenabout 1-100 mg/m², preferably about 2.5-50 mg/m², more preferably about3-40 mg/m², most preferably 7.5-30 mg/m². In a particular embodiment thedosage is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/m² of HMN-214 (or HMN-176)administered to a subject once, twice, three times or more than threetimes a week, for example, on days 1-21 of a 28 day cycle. In anotherparticular embodiment the dosage is 10 to 48 mg/m² preferably 18 to 30mg/m² of HMN-214 (or HMN-176) administered once, twice, three times ormore than three times a week, for example, days 1-5 of a 28 day cycle.In some embodiments, HMN-214 (or HMN-176) is administered orally.

Rigosertib (ON-01910)

The benzyl styryl sulfone analogue ON 01910 is an ATP-noncompetitive,multitargeted inhibitor of several tyrosine kinases and cyclin-dependentkinase 1 (Cdk1; IC50=18-260 nmol/L). It is reported to have aparticularly strong potency (IC50=9-10 nmol/L) toward Plk1 (Gumireddy,et al., Cancer Cell, 7:275-86 (2005)). The structure of ON-01910 is

In preclinical animal studies in mouse xenograft models of Bel-7402,MCF-7, and MIA-PaCa cells, Rigosertib (250 mg/kg) inhibited tumor growthand (200 mg/kg) showed inhibition of tumor growth in a mouse xenograftmodel of BT20 cells (Gumireddy, et al., Cancer Cell, 7:275-86 (2005),Reddy, et al., J. Med Chem., 54(18), 6254-6276 (2011)).

A phase I first-in-humans study of ON 01910 was conducted in 20 patientswith advanced solid tumors (none with NSCLC). The agent was administeredi.v. at 80 to 4,370 mg by accelerated titration design on days 1, 4, 8,11, 15, and 18 in 28-day cycles (Jimeno, et al., J. Clin. Oncol.,26:5504-10 (2008). Grade 3 abdominal pain was reported as a DLT at anMTD of 3,120 mg.

In a clinical trial testing the safety and pharmacokinetics of oral on01910 in patients with myelodysplastic syndrome, ON 01910 was giventwice a day up to 14 days at doses escalating from 70 mg to 700 mg.

Therefore, in some embodiments, ON 01910 is administered to a subject 1,2, 3 or more days a week in a dosage of about 50-6,000 mg, preferablyabout 60-4,500 mg, more preferably about 150 mg-1,500 mg once daily, or75-750 mg twice daily. In particular embodiments, ON 01910 administeredto a subject once, twice, three times or more than three times a week,or once every two, three or four weeks. In a specific embodiment, thedrug is administered every day for 14 days. In some embodiments, ON01910 is administered by intravenous injection or infusion.

MLN0905

MLN0905 is a potent inhibitor of PLK1 with IC50 of 2 nM. MLN0905inhibits cell mitosis with EC50 of 9 nM and Cdc25C-T96 phosphorylation,a direct readout of PLK1 inhibition, with EC50 of 29 nM (Duffey, MedChem, 55(1), 197-208 (2012)). The structure of MLN0905 is

In preclinical experiments indicate an effective dosage range of about 1mg/kg-50 mg/kg. One study indicates a preferred dosage of about 3-15mg/kg with a maximum tolerated dose on QD (daily) schedule to be 6.25mg/kg and on the QD×3/wk (3-days on/4-days off) schedule to be 14.5mg/kg (Shi, et al., Mol. Cancer Thera., 11(9), 2045-2053 (2012)).

RO3280

RO3280 is a potent, highly selective inhibitor of Polo-like kinase 1(PLK1) with IC50 of 3 nM. The structure of RO3280 is

RO3280 shows the strong anti-proliferative activity against lung cancercell line H82, colorectal cancer cell HT-29, breast cancer cellMDA-MB-468, prostate cancer cell PC3 and skin cancer cell A375 withIC50s of 5, 10, 19, 12 and 70 nM, respectively. RO3280 also showedpromising antitumor activity in nude mouse implanted with HT-29 humancolorectal tumors ranging from 72% tumor growth inhibition when dosedonce weekly at 40 mg/kg, to complete tumor regression when dosed morefrequently (Chen, et al., Bioorg. Med Chem. Lett., 22(2), 1247-1250(2012).

NMS-1286937

NMS 1286937, also known as NMS-P937, is an orally bioavailable,small-molecule Polo-like kinase 1 (PLK1) inhibitor with potentialantineoplastic activity. Preclinical evaluation has shown high potencyof the compound in proliferation assays, displaying low nanomolaractivity on a large number of cell lines, representative of both solidand hematological tumors. A phase 1 dose escalation study of nms-1286937administered to adult patients with advanced/metastatic solid tumors hasbeen completed (Hartsink-Segers, et al., Haematologica. 98(10):1539-46(2013)).

TAK-960

TAK-960 is an orally bioavailable, potent, and selective PLK1 inhibitorthat has shown activity in several tumor cell lines, including thosethat express multidrug-resistant protein 1 (MDRI) (Hikichi, et al., MolCancer Ther. 11(3):700-9 (2012)). A Phase 1, open-label, dose-escalationstudy of orally administered TAK-960 has been completed.

CFI-400945 Fumarate

CFI-400945 is an inhibitor of polo-like kinase 4 (PLK4). Many tumors areshown to make too much PLK4. Phase 1 clinical trials of CFI-400945fumarate delivered orally, at dose levels of 3, 6, 11, 16, 24, and 32mg/day are currently underway (Mason, et al., Cancer Cell, V 26(2), pp.163-176 (2014)).

b. Functional Nucleic Acid Inhibitors of PLK

In some embodiments, the polo-like kinase inhibitor is a functionalnucleic acid that targets Plk1, Plk2/Snk, Plk3/Prk/FnK, Plk4/Sak, orPlk5. The functional nucleic acid can be, for example, an antisensemolecule, aptamer, ribozyme, triplex forming oligonucleotide, externalguide sequence, or RNAi that targets inhibits or reduces expression ortranslation of Plk1, Plk2/Snk, Plk3/Prk/FnK, Plk4/Sak, or Plk5 mRNA.

TKM-080301

In a particular embodiment, the Functional Nucleic Acid Inhibitor of PLKis TKM-080301. TKM-080301 has been effective when given in a 30-minuteintravenous infusion. Phase clinical trials are currently underway,including doses ranging from 0.15 mg/kg per week to 0.9 mg/kg per week.Dose-limiting toxicities were observed at the 0.9 mg/kg per-week.

3. Additional Active Agents

In some embodiments, the combination therapy includes additional activeagents. In addition to one or more antiandrogen or androgen antagonist,and one or more Plk inhibitors, the combination therapies can includeany of the additional agents or components discussed herein, or known inthe art to be coadministered with an antiandrogen or androgenantagonist, or with a Plk inhibitor. For example, abiraterone acetate isroutinely administered in combination with a steroid such as prednisoneor prednisolone. Therefore, in some embodiments, the combinationincludes prednisone or prednisolone.

In some embodiments, the combination therapy includes administration ofan additional antiandrogen therapeutic agent, an immunotherapeuticagent, an agent for treating bone complications, a chemotherapeuticagent, or a combination thereof. In some embodiments, the combinationtherapy includes administration of an additional first or second linetherapeutic agent for treatment of CRPC, such as one or more of theagents reviewed in Shapiro and Tareen, Expert Rev. Anticancer Ther.,12(7):951-964 (2012), Heidegger, et al., J. Steroid Biochem. Mol. Biol.,138(100): 248-256 (2013), and Lui, et al., Cancer Control, 20(3):181-187(2013), each of which is specifically incorporated by reference hereinin its entirety.

In some embodiments, the additional active agent is administered to thesubject during the same cycle as the antiandrogen or androgen antagonistand Plk inhibitor. For example, the combination therapy can include aPlk inhibitor, abiraterone and a second antiandrogen or androgenantagonist. In a particular embodiment, the second antiandrogen orandrogen antagonist is enzalutamide which is an androgen receptorinhibitor (Efstathiou, et al., European Cancer Congress 2013, Sep. 27-1,2013, Amsterdam, The Netherlands, Abstract 2854).

It is appreciated that some combinations should not be administeredsimultaneously. Such combinations are preferably administered in series,for example using cycles, or drug holidays. For example, sipuleucel-Tsold under the trade name PROVENGE® is an immune therapy that stimulatesthat immune system to attack the cancer. It is typically not advised tosimultaneously administer abiraterone acetate and sipuleucel-T becauseabiraterone is typically administered simultaneously with a steroid.However, sequential administration of sipuleucel-T and abirateroneacetate has been proposed.

B. Formulations

Formulations of and pharmaceutical compositions including one or moreactive agents are provided. The combination therapies can includeadministration of the active agents together in the same admixture, orin separate admixtures. Therefore, the pharmaceutical compositions caninclude an antiandrogen or androgen antagonist, Plk inhibitor, or acombination thereof. In some embodiments, the pharmaceuticalcompositions can include one or more additional active agents.Therefore, in some embodiments, the pharmaceutical composition includestwo, three, or more active agents. The pharmaceutical compositions canbe formulated as a pharmaceutical dosage unit, referred to as a unitdosage form. Such formulations typically include an effective amount ofan antiandrogen or androgen antagonist, a Plk inhibitor, or acombination thereof. Effective amounts of the disclosed active agentsare discussed in more detail below. It will be appreciated that in someembodiments the effective amount of antiandrogen or androgen antagonist,or Plk inhibitor in a combination therapy is different from that amountthat would be effective for the antiandrogen or androgen antagonist, orPlk inhibitor to achieve the same result individually. For example, insome embodiments the effective amount of antiandrogen or androgenantagonist, or Plk inhibitor, is a lower dosage of the antiandrogen orandrogen antagonist, or Plk inhibitor in a combination therapy than thedosage of the antiandrogen or androgen antagonist, or Plk inhibitor thatis effective when one agent is administered without the other.Alternatively, in some embodiments the effective amount of antiandrogenor androgen antagonist, or Plk inhibitor, is a higher dosage of theantiandrogen or androgen antagonist, or Plk inhibitor in a combinationtherapy than the dosage of the antiandrogen or androgen antagonist, orPlk inhibitor that is effective when one agent is administered withoutthe other. In other embodiments, the dosage of one agent is higher andthe dosage of the other agent is lower than one agent is administeredwithout the other. In some case, the agents are not effective whenadministered alone, and only effective when administered in combination.

1. Delivery Vehicles

The active agents can be administered and taken up into the cells of asubject with or without the aid of a delivery vehicle. Appropriatedelivery vehicles for the disclosed active agents are known in the artand can be selected to suit the particular inhibitor. For example, insome embodiments, the active agent(s) is incorporated into orencapsulated by a nanoparticle, microparticle, micelle, syntheticlipoprotein particle, or carbon nanotube. For example, the compositionscan be incorporated into a vehicle such as polymeric microparticleswhich provide controlled release of the active agent(s). In someembodiments, release of the drug(s) is controlled by diffusion of theactive agent(s) out of the microparticles and/or degradation of thepolymeric particles by hydrolysis and/or enzymatic degradation. Suitablepolymers include ethylcellulose and other natural or synthetic cellulosederivatives. Polymers which are slowly soluble and form a gel in anaqueous environment, such as hydroxypropyl methylcellulose orpolyethylene oxide may also be suitable as materials for drug containingmicroparticles. Other polymers include, but are not limited to,polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such aspolylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide)(PLGA), poly-3-hydroxybut rate (PHB) and copolymers thereof,poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactoneand copolymers thereof, and combinations thereof. In some embodiments,both agents are incorporated into the same particles and are formulatedfor release at different times and/or over different time periods. Forexample, in some embodiments, one of the agents is released entirelyfrom the particles before release of the second agent begins. In otherembodiments, release of the first agent begins followed by release ofthe second agent before the all of the first agent is released. In stillother embodiments, both agents are released at the same time over thesame period of time or over different periods of time.

The active agent(s) can be incorporated into a delivery vehicle preparedfrom materials which are insoluble in aqueous solution or slowly solublein aqueous solution, but are capable of degrading within the GI tract bymeans including enzymatic degradation, surfactant action of bile acids,and/or mechanical erosion. As used herein, the term “slowly soluble inwater” refers to materials that are not dissolved in water within aperiod of 30 minutes. Preferred examples include fats, fatty substances,waxes, wax-like substances and mixtures thereof. Suitable fats and fattysubstances include fatty alcohols (such as lauryl, myristyl stearyl,cetyl or cetostearyl alcohol), fatty acids and derivatives, including,but not limited to, fatty acid esters, fatty acid glycerides (mono-, di-and tri-glycerides), and hydrogenated fats. Specific examples include,but are not limited to hydrogenated vegetable oil, hydrogenatedcottonseed oil, hydrogenated castor oil, hydrogenated oils availableunder the trade name Sterotex®, stearic acid, cocoa butter, and stearylalcohol. Suitable waxes and wax-like materials include natural orsynthetic waxes, hydrocarbons, and normal waxes.

Specific examples of waxes include beeswax, glycowax, castor wax,carnauba wax, paraffins and candelilla wax. As used herein, a wax-likematerial is defined as any material which is normally solid at roomtemperature and has a melting point of from about 30 to 300° C. Therelease point and/or period of release can be varied as discussed above.

2. Pharmaceutical Compositions

Pharmaceutical compositions including active agent(s) with or without adelivery vehicle are provided. Pharmaceutical compositions can be foradministration by parenteral (intramuscular, intraperitoneal,intravenous (IV) or subcutaneous injection), enteral, or transmucosal(nasal, vaginal, rectal, or sublingual) routes of administration orusing bioerodible inserts and can be formulated in dosage formsappropriate for each route of administration.

In certain embodiments, the compositions are administered locally, forexample, by injection directly into a site to be treated (e.g., into atumor). In some embodiments, the compositions are injected or otherwiseadministered directly into the vasculature onto vascular tissue at oradjacent to the intended site of treatment (e.g., adjacent to a tumor).Typically, local administration causes an increased localizedconcentration of the compositions which is greater than that which canbe achieved by systemic administration. Targeting of the molecules orformulation can be used to achieve more selective delivery.

a. Formulations for Parenteral Administration

Active agent(s) and pharmaceutical compositions thereof can beadministered in an aqueous solution, by parenteral injection. Theformulation may also be in the form of a suspension or emulsion. Ingeneral, pharmaceutical compositions are provided including effectiveamounts of the active agent(s) and optionally include pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers. Such compositions include diluents sterile water,buffered saline of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; and optionally, additives such asdetergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80 alsoreferred to as polysorbate 20 or 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol). Examples ofnon-aqueous solvents or vehicles are propylene glycol, polyethyleneglycol, vegetable oils, such as olive oil and corn oil, gelatin, andinjectable organic esters such as ethyl oleate. The formulations may belyophilized and redissolved/resuspended immediately before use. Theformulation may be sterilized by, for example, filtration through abacteria retaining filter, by incorporating sterilizing agents into thecompositions, by irradiating the compositions, or by heating thecompositions.

b. Enteral Formulations

Suitable oral dosage forms include tablets, capsules, solutions,suspensions, syrups, and lozenges. Tablets can be made using compressionor molding techniques well known in the art. Gelatin or non-gelatincapsules can prepared as hard or soft capsule shells, which canencapsulate liquid, solid, and semi-solid fill materials, usingtechniques well known in the art. Formulations may be prepared using apharmaceutically acceptable carrier. As generally used herein “carrier”includes, but is not limited to, diluents, preservatives, binders,lubricants, disintegrators, swelling agents, fillers, stabilizers, andcombinations thereof.

Carrier also includes all components of the coating composition, whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants. Delayed release dosage formulations may be prepared asdescribed in standard references. These references provide informationon carriers, materials, equipment and process for preparing tablets andcapsules and delayed release dosage forms of tablets, capsules, andgranules.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients include, but are notlimited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants. Diluents, also referred to as “fillers,”are typically necessary to increase the bulk of a solid dosage form sothat a practical size is provided for compression of tablets orformation of beads and granules. Suitable diluents include, but are notlimited to, dicalcium phosphate dihydrate, calcium sulfate, lactose,sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose,kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinizedstarch, silicone dioxide, titanium oxide, magnesium aluminum silicateand powdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone® XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard drug decomposition reactions,which include, by way of example, oxidative reactions. Suitablestabilizers include, but are not limited to, antioxidants, butylatedhydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E,tocopherol and its salts; sulfites such as sodium metabisulphite;cysteine and its derivatives; citric acid; propyl gallate, and butylatedhydroxyanisole (BHA).

Oral dosage forms, such as capsules, tablets, solutions, andsuspensions, can for formulated for controlled release. For example, theone or more compounds and optional one or more additional active agentscan be formulated into nanoparticles, microparticles, and combinationsthereof, and encapsulated in a soft or hard gelatin or non-gelatincapsule or dispersed in a dispersing medium to form an oral suspensionor syrup. The particles can be formed of the drug and a controlledrelease polymer or matrix. Alternatively, the drug particles can becoated with one or more controlled release coatings prior toincorporation in to the finished dosage form.

In another embodiment, the one or more compounds and optional one ormore additional active agents are dispersed in a matrix material, whichgels or emulsifies upon contact with an aqueous medium, such asphysiological fluids. In the case of gels, the matrix swells entrappingthe active agents, which are released slowly over time by diffusionand/or degradation of the matrix material. Such matrices can beformulated as tablets or as fill materials for hard and soft capsules.

In still another embodiment, the one or more compounds, and optional oneor more additional active agents are formulated into a sold oral dosageform, such as a tablet or capsule, and the solid dosage form is coatedwith one or more controlled release coatings, such as a delayed releasecoatings or extended release coatings. The coating or coatings may alsocontain the compounds and/or additional active agents.

The extended release formulations are generally prepared as diffusion orosmotic systems, which are known in the art. A diffusion systemtypically consists of two types of devices, a reservoir and a matrix,and is well known and described in the art. The matrix devices aregenerally prepared by compressing the drug with a slowly dissolvingpolymer carrier into a tablet form. The three major types of materialsused in the preparation of matrix devices are insoluble plastics,hydrophilic polymers, and fatty compounds. Plastic matrices include, butare not limited to, methyl acrylate-methyl methacrylate, polyvinylchloride, and polyethylene. Hydrophilic polymers include, but are notlimited to, cellulosic polymers such as methyl and ethyl cellulose,hydroxyalkylcelluloses such as hydroxypropyl-cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, andCarbopol® 934, polyethylene oxides and mixtures thereof. Fatty compoundsinclude, but are not limited to, various waxes such as carnauba wax andglyceryl tristearate and wax-type substances including hydrogenatedcastor oil or hydrogenated vegetable oil, or mixtures thereof.

Alternatively, extended release formulations can be prepared usingosmotic systems or by applying a semi-permeable coating to the dosageform. In the latter case, the desired drug release profile can beachieved by combining low permeable and high permeable coating materialsin suitable proportion.

The devices with different drug release mechanisms described above canbe combined in a final dosage form including single or multiple units.Examples of multiple units include, but are not limited to, multilayertablets and capsules containing tablets, beads, or granules. Animmediate release portion can be added to the extended release system bymeans of either applying an immediate release layer on top of theextended release core using a coating or compression process or in amultiple unit system such as a capsule containing extended and immediaterelease beads.

Extended release tablets containing hydrophilic polymers are prepared bytechniques commonly known in the art such as direct compression, wetgranulation, or dry granulation. Their formulations usually incorporatepolymers, diluents, binders, and lubricants as well as the activepharmaceutical ingredient. The usual diluents include inert powderedsubstances such as starches, powdered cellulose, especially crystallineand microcrystalline cellulose, sugars such as fructose, mannitol andsucrose, grain flours and similar edible powders. Typical diluentsinclude, for example, various types of starch, lactose, mannitol,kaolin, calcium phosphate or sulfate, inorganic salts such as sodiumchloride and powdered sugar. Powdered cellulose derivatives are alsouseful. Typical tablet binders include substances such as starch,gelatin and sugars such as lactose, fructose, and glucose. Natural andsynthetic gums, including acacia, alginates, methylcellulose, andpolyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilicpolymers, ethylcellulose and waxes can also serve as binders. Alubricant is necessary in a tablet formulation to prevent the tablet andpunches from sticking in the die. The lubricant is chosen from suchslippery solids as talc, magnesium and calcium stearate, stearic acidand hydrogenated vegetable oils.

Extended release tablets containing wax materials are generally preparedusing methods known in the art such as a direct blend method, acongealing method, and an aqueous dispersion method. In the congealingmethod, the drug is mixed with a wax material and either spray-congealedor congealed and screened and processed.

Delayed release formulations can be created by coating a solid dosageform with a polymer film, which is insoluble in the acidic environmentof the stomach, and soluble in the neutral environment of the smallintestine.

The delayed release dosage units can be prepared, for example, bycoating a drug or a drug-containing composition with a selected coatingmaterial. The drug-containing composition may be, e.g., a tablet forincorporation into a capsule, a tablet for use as an inner core in a“coated core” dosage form, or a plurality of drug-containing beads,particles or granules, for incorporation into either a tablet orcapsule. Preferred coating materials include bioerodible, graduallyhydrolyzable, gradually water-soluble, and/or enzymatically degradablepolymers, and may be conventional “enteric” polymers. Enteric polymers,as will be appreciated by those skilled in the art, become soluble inthe higher pH environment of the lower gastrointestinal tract or slowlyerode as the dosage form passes through the gastrointestinal tract,while enzymatically degradable polymers are degraded by bacterialenzymes present in the lower gastrointestinal tract, particularly in thecolon. Suitable coating materials for effecting delayed release include,but are not limited to, cellulosic polymers such as hydroxypropylcellulose, hydroxyethyl cellulose, hydroxymethyl cellulose,hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, hydroxypropylmethyl cellulose phthalate, methylcellulose,ethyl cellulose, cellulose acetate, cellulose acetate phthalate,cellulose acetate trimellitate and carboxymethylcellulose sodium;acrylic acid polymers and copolymers, preferably formed from acrylicacid, methacrylic acid, methyl acrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate, and other methacrylic resinsthat are commercially available under the tradename Eudragit® (RohmPharma; Westerstadt, Germany), including EUDRAGIT® L30D-55 and L100-55(soluble at pH 5.5 and above), EUDRAGIT® L-100 (soluble at pH 6.0 andabove), EUDRAGIT® S (soluble at pH 7.0 and above, as a result of ahigher degree of esterification), and EUDRAGITS® NE, RL and RS(water-insoluble polymers having different degrees of permeability andexpandability); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetatecrotonic acid copolymer, and ethylene-vinyl acetate copolymer;enzymatically degradable polymers such as azo polymers, pectin,chitosan, amylose and guar gum; zein and shellac. Combinations ofdifferent coating materials may also be used. Multi-layer coatings usingdifferent polymers may also be applied.

The preferred coating weights for particular coating materials may bereadily determined by those skilled in the art by evaluating individualrelease profiles for tablets, beads and granules prepared with differentquantities of various coating materials. It is the combination ofmaterials, method and form of application that produce the desiredrelease characteristics, which one can determine only from the clinicalstudies.

The coating composition may include conventional additives, such asplasticizers, pigments, colorants, stabilizing agents, glidants, etc. Aplasticizer is normally present to reduce the fragility of the coating,and will generally represent about 10 wt. % to 50 wt. % relative to thedry weight of the polymer. Examples of typical plasticizers includepolyethylene glycol, propylene glycol, triacetin, dimethyl phthalate,diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethylcitrate, tributyl citrate, triethyl acetyl citrate, castor oil andacetylated monoglycerides. A stabilizing agent is preferably used tostabilize particles in the dispersion. Typical stabilizing agents arenonionic emulsifiers such as sorbitan esters, polysorbates andpolyvinylpyrrolidone. Glidants are recommended to reduce stickingeffects during film formation and drying, and will generally representapproximately 25 wt. % to 100 wt. % of the polymer weight in the coatingsolution. One effective glidant is talc. Other glidants such asmagnesium stearate and glycerol monostearates may also be used. Pigmentssuch as titanium dioxide may also be used. Small quantities of ananti-foaming agent, such as a silicone (e.g., simethicone), may also beadded to the coating composition.

Kinase inhibitor and compositions thereof can be applied formulated forpulmonary or mucosal administration. The administration can includedelivery of the composition to the lungs, nasal, oral (sublingual,buccal), vaginal, or rectal mucosa.

In one embodiment, the compounds are formulated for pulmonary delivery,such as intranasal administration or oral inhalation. The respiratorytract is the structure involved in the exchange of gases between theatmosphere and the blood stream. Pulmonary administration of therapeuticcompositions including low molecular weight drugs has been observed, forexample, beta-androgenic antagonists to treat asthma. Other therapeuticagents that are active in the lungs have been administered systemicallyand targeted via pulmonary absorption. The term aerosol as used hereinrefers to any preparation of a fine mist of particles, which can be insolution or a suspension, whether or not it is produced using apropellant. Aerosols can be produced using standard techniques, such asultrasonication or high-pressure treatment.

Carriers for pulmonary formulations can be divided into those for drypowder formulations and for administration as solutions. Aerosols forthe delivery of therapeutic agents to the respiratory tract are known inthe art. For administration via the upper respiratory tract, theformulation can be formulated into a solution, e.g., water or isotonicsaline, buffered or un-buffered, or as a suspension, for intranasaladministration as drops or as a spray. Preferably, such solutions orsuspensions are isotonic relative to nasal secretions and of about thesame pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0to pH 7.0. Buffers should be physiologically compatible and include,simply by way of example, phosphate buffers. For example, arepresentative nasal decongestant is described as being buffered to a pHof about 6.2. One skilled in the art can readily determine a suitablesaline content and pH for an innocuous aqueous solution for nasal and/orupper respiratory administration.

Preferably, the aqueous solution is water, physiologically acceptableaqueous solutions containing salts and/or buffers, such as phosphatebuffered saline (PBS), or any other aqueous solution acceptable foradministration to an animal or human. Such solutions are well known to aperson skilled in the art and include, but are not limited to, distilledwater, de-ionized water, pure or ultrapure water, saline,phosphate-buffered saline (PBS). Other suitable aqueous vehiclesinclude, but are not limited to, Ringer's solution and isotonic sodiumchloride. Aqueous suspensions may include suspending agents such ascellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gumtragacanth, and a wetting agent such as lecithin. Suitable preservativesfor aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

In another embodiment, solvents that are low toxicity organic (i.e.,non-aqueous) class 3 residual solvents, such as ethanol, acetone, ethylacetate, tetrahydrofuran, ethyl ether, and propanol may be used for theformulations. The solvent is selected based on its ability to readilyaerosolize the formulation. The solvent should not detrimentally reactwith the compounds. An appropriate solvent should be used that dissolvesthe compounds or forms a suspension of the compounds. The solvent shouldbe sufficiently volatile to enable formation of an aerosol of thesolution or suspension. Additional solvents or aerosolizing agents, suchas freons, can be added as desired to increase the volatility of thesolution or suspension.

In one embodiment, compositions may contain minor amounts of polymers,surfactants, or other excipients well known to those of the art. In thiscontext, “minor amounts” means no excipients are present that mightaffect or mediate uptake of the compounds in the lungs and that theexcipients that are present are present in amount that do not adverselyaffect uptake of compounds in the lungs.

Dry lipid powders can be directly dispersed in ethanol because of theirhydrophobic character. For lipids stored in organic solvents such aschloroform, the desired quantity of solution is placed in a vial, andthe chloroform is evaporated under a stream of nitrogen to form a drythin film on the surface of a glass vial. The film swells easily whenreconstituted with ethanol. To fully disperse the lipid molecules in theorganic solvent, the suspension is sonicated. Nonaqueous suspensions oflipids can also be prepared in absolute ethanol using a reusable PARI LCJet+ nebulizer (PARI Respiratory Equipment, Monterey, Calif.).

Dry powder formulations (“DPFs”) with large particle size have improvedflowability characteristics, such as less aggregation, easieraerosolization, and potentially less phagocytosis. Dry powder aerosolsfor inhalation therapy are generally produced with mean diametersprimarily in the range of less than 5 microns, although a preferredrange is between one and ten microns in aerodynamic diameter. Large“carrier” particles (containing no drug) have been co-delivered withtherapeutic aerosols to aid in achieving efficient aerosolization amongother possible benefits.

Polymeric particles may be prepared using single and double emulsionsolvent evaporation, spray drying, solvent extraction, solventevaporation, phase separation, simple and complex coacervation,interfacial polymerization, and other methods well known to those ofordinary skill in the art. Particles may be made using methods formaking microspheres or microcapsules known in the art. The preferredmethods of manufacture are by spray drying and freeze drying, whichentails using a solution containing the surfactant, spraying to formdroplets of the desired size, and removing the solvent.

Formulations for pulmonary delivery include unilamellar phospholipidvesicles, liposomes, or lipoprotein particles. Formulations and methodsof making such formulations containing nucleic acid are well known toone of ordinary skill in the art. Liposomes are formed from commerciallyavailable phospholipids supplied by a variety of vendors includingAvanti Polar Lipids, Inc. (Birmingham, Ala.). In one embodiment, theliposome can include a ligand molecule specific for a receptor on thesurface of the target cell to direct the liposome to the target cell.

C. Adjunct and Additional Therapies and Procedures

The combination therapies can be administered to a subject incombination with one or more adjunct therapies or procedures, or can bean adjunct therapy to one or more primary therapies or producers. Theadditional therapy or procedure can be simultaneous or sequential withthe combination therapy. In some embodiment the additional therapy isperformed between drug cycles or during a drug holiday that is part ofthe combination therapy dosage regime. In preferred embodiment, theadditional therapy is a conventional treatment for cancer, morepreferably a conventional treatment for prostate or breast cancer, mostpreferably a conventional treatment for hormone-resistant prostate orbreast cancer. For example, in some embodiments, the additional therapyor procedure is surgery, a radiation therapy, or chemotherapy. Forexample, in a particular embodiment, combination therapies usedsimultaneously or sequentially with a regime of a chemotherapeuticagent, e.g., docetaxel or cabazitaxel. As discussed in more detailbelow, in some embodiment, the adjunct or additional therapy is part ofthe combination therapy.

III. Methods of Treatment

It has been established that androgen receptor antagonists oranti-androgens can be used in combination with inhibitors of Plk toprovide enhanced antitumor activity as compared to the use of eitheragent alone. It is believed that inhibition of androgen receptorsignaling by an androgen receptor antagonist or anti-androgen gives riseto an increased expression of genes involved in the retinoic acidreceptor (RA) signaling pathway. Up-regulation of RA signaling isbelieved to enhance the sensitivity of cancer cells to Plk inhibitoractivity. Further, the combined effects of RA up-regulation and Plkinhibitor activity are believed to inhibit or reduce the activity of theNrf 1/2 (nuclear respiratory factor 1/2) and/or Elk1 transcriptionfactors, and thereby reduce or inhibit the expression of genes that isdriven by the Nrf1/2 or Elk1 transcription factors. Motifs bound by ELK1and NRF1 positively correlate with malignant progression of breastcancer, colorectal tumors, Hepatoma and thyroid oncocytoma.

Methods of treating one or more symptoms of cancer in a subject areprovided. In certain embodiments, the methods include administering to asubject with cancer an effective amount of an androgen receptorantagonist or anti-androgen, or a derivative, analog or prodrug, or apharmacologically active salt thereof in combination with one or moreinhibitors of Plk to reduce or inhibit one or more symptoms of thecancer. In preferred embodiments, the androgen receptor antagonist oranti-androgen and inhibitors of Plk can be used in combination toprovide enhanced antitumor activity as compared to the use of eitheragent alone. The methods can include contacting one or more cancer cellsexpressing the androgen receptor and polo-like kinase with an effectiveamount of an androgen receptor antagonist or anti-androgen incombination with one or more inhibitors of Plk to decrease or inhibitthe proliferation and/or viability of the cancer cells compared tountreated control cancer cells. In certain embodiments the methods aremore effective in treating cancer in a subject having cancer cells thatexhibit expression of one or more genes involved in the retinoic acidsignaling pathway. In preferred embodiments, the cancer cells exhibitincreased retinoic acid signaling relative to non-cancer cells. Thegenes involved in retinoic acid signaling can include Retinoic AcidReceptor Alpha (RARA); Retinoic Acid Receptor Gamma (RARG); RetinolDehydrogenase (ADH4); and Retinaldehyde Reductase (DHRS3). In otherembodiments, the methods are more effective in treating cancer in asubject having cancer cells that exhibit increased expression of one ormore genes that contain binding sites for the transcription factorsNrf1/2 and/or Elk1 adjacent to their transcription start sites inresponse to combination treatment with an anti-androgen or androgenantagonist plus a Plk inhibitor.

The antiandrogen or androgen antagonist and Plk inhibitor can beadministered locally or systemically to the subject, or coated orincorporated onto, or into a device.

A. Methods for Selecting Patients for Androgen Antagonist and PlkInhibitor Combination Therapies

Methods for characterizing tumors and/or for characterizing the tumormicroenvironment are provided. In particular, the disclosure pertains tomethods for characterizing tumors so as to assess the extent to whichthe tumor cells and/or tumor infiltrating cells or tumor associatedcells express genes associated with sensitivity to combination therapywith androgen receptor antagonist or anti-androgen, in combination withone or more inhibitors of Plk. For example, tumor cells and/or tumorinfiltrating cells or tumor associated cells that are sensitive to morethan additive effects of combination therapy with androgen receptorantagonist or anti-androgen in combination with one or more inhibitorsof Plk can express genes involved in the Retinoic Acid Receptor (RA)signaling pathway prior to treatment, and/or show up-regulation of genesdriven by the NRF1/2 or Elk1 transcription factors after combinationtreatment. The disclosure concerns the uses of such methods in thediagnosis and the treatment of cancer and other diseases. Therefore,methods for characterizing a cell of a tumor are provided. The methodscan include determining whether a cell of the tumor expresses one ormore of the components of the Retinoic Acid Receptor (RA) signalingpathway, specifically genes involved in retinoic acid signaling,including Retinoic Acid Receptor Alpha (RARA); Retinoic Acid ReceptorGamma (RARG); Retinol Dehydrogenase (ADH4); and Retinaldehyde Reductase(DHRS3).

Methods for assessing the amenability of subject to a proposedanti-cancer therapy are also provided. The methods can include, forexample, characterizing cells of a tumor of the subject by determiningwhether the cells of the tumor express genes that are associated withsensitivity to more than additive effects of androgen receptorantagonist or anti-androgen in combination with one or more inhibitorsof Plk. For example, subjects having cancer cells that express genesinvolved in the Retinoic Acid Receptor (RA) signaling pathway can beselected for treatment with an androgen receptor antagonist oranti-androgen in combination with one or more inhibitors of Plk. Methodsfor assessing the efficacy of an anti-cancer therapy provided to asubject are also disclosed. The methods can include, for example,characterizing cells of a tumor of the patient during the course of thetherapy or after the completion thereof, wherein said characterizationcan include determining whether the cells of the tumor express genesinvolved in the Retinoic Acid Receptor (RA) signaling pathway, or showincreased expression of genes that are driven by the Nrf1/2 or Elk1transcription factors in response to the treatment.

Methods for selecting patients for anti-cancer therapy based oncharacterization of the tumor or tumor microenvironment are alsoprovided. In some embodiments, cancer patient tumor samples arecharacterized prior to and following treatment with following specificchemotherapeutic and/or biologic therapies, and/or other therapeuticinterventions (e.g. radiation, cryoablation, surgical resection of thetumor, etc.) in order to see if the expression patterns of genesinvolved in the Retinoic Acid Receptor (RA) signaling pathway, or genesthat are driven by the Nrf1/2 or Elk1 transcription factors, within thetumor microenvironment have changed.

The methods typically include detecting genes involved in the RetinoicAcid Receptor (RA) signaling pathway, or genes that are driven by theNrf1/2 or Elk1 transcription factors, alone or in combination with oneor more other biomarkers of cancer cells. Suitable methods of detectionare known in the art. For example, some of the disclosed methods includea step of contacting the cell of the tumor with a molecule thatimmunospecifically or physiospecifically binds proteins involved in theRetinoic Acid Receptor (RA) signaling pathway, or examining RNAexpression for genes regulated by the Nrf1/2 or Elk1 transcriptionfactors using qPCR, microarray methods, or RNA-Seq. In some preferredembodiments, the molecule that immunospecifically or physiospecificallybinds proteins involved in the Retinoic Acid Receptor (RA) signalingpathway is an antibody or an antigen-binding fragment thereof.

B. Methods of Administration and Dosage Regimes

The combination therapies and treatment regimens typically includetreatment of a disease or symptom thereof, or a method for achieving adesired physiological change, including administering to an animal, suchas a mammal, especially a human being, an effective amount of anantiandrogen or androgen antagonist and a Plk inhibitor to treat thedisease or symptom thereof, or to produce the physiological change,wherein the chemical agents or components are administered together,such as part of the same composition, or administered separately andindependently at the same time or at different times (i.e.,administration of the antiandrogen or androgen antagonist and the Plkinhibitor is separated by a finite period of time from each other).Therefore, the term “combination” or “combined” is used to refer toeither concomitant, simultaneous, or sequential administration of theantiandrogen or androgen antagonist and the Plk inhibitor. Thecombinations can be administered either concomitantly (e.g., as anadmixture), separately but simultaneously (e.g., via separateintravenous lines into the same subject; one agent is given orally whilethe other agent is given by infusion or injection, etc.,), orsequentially (e.g., one agent is given first followed by the second).

When used for treating cancer, the amount of antiandrogen or androgenantagonist present in a pharmaceutical dosage unit, or otherwiseadministered to a subject can be the amount effective to reduce theproliferation, viability, or a combination thereof of the cancer cellswhen administered in combination with a Plk inhibitor. Likewise, theamount of Plk inhibitor present in a pharmaceutical dosage unit, orotherwise administered to a subject can be the amount effective toreduce the proliferation, viability, or a combination thereof of thecancer cells when administered in combination with an antiandrogen orandrogen antagonist. Therefore, in some embodiments the amount of theactive agents is effective to reduce, slow or halt tumor progression, toreduce tumor burden, or a combination thereof. In some embodiment, theamount of the active agents is effective to alter a measurablebiochemical or physiological marker. For example, if the cancer isprostate cancer, the amount of the active agents can be effective toreduce the level of prostate specific antigen (PSA) concentration in theblood compared to the PSA concentration prior to treatment. In someembodiments, the active agents are administered in an effective amountto reduce or prevent cancer progression despite a rise in androgenlevels or PSA concentration in levels.

In preferred embodiments, administration of the antiandrogen or androgenantagonist and the Plk inhibitor achieves a result greater than when theantiandrogen or androgen antagonist and the Plk inhibitor areadministered alone or in isolation. For example, in some embodiments,the result achieved by the combination is partially or completelyadditive of the results achieved by the individual components alone. Inthe most preferred embodiments, the result achieved by the combinationis more than additive of the results achieved by the individualcomponents alone. In some embodiments, the effective amount of one orboth agents used in combination is lower than the effective amount ofeach agent when administered separately. In some embodiments, the amountof one or both agents when used in the combination therapy issub-therapeutic when used alone.

The effect of the combination therapy, or individual agents thereof candepend on the disease or condition to be treated or progression thereof.For example, as illustrated in the Examples below, an agent such isabiraterone can be used a first or second line therapy for treatment ofprostate cancer. However, over time, the cancer can develop a resistanceto abiraterone. Subsequent treatment of the cancer with abiraterone incombination with a Plk inhibitor such as BI2536 “re-sensitizes” thecancer to abiraterone treatment. Accordingly, in some embodiments, theeffect of the combination on a cancer can compared to the effect of theindividual agents alone on the cancer.

The Examples illustrate that the effect of the combination therapies canbe hormone independent (e.g., is still effective in the presence ofrising levels of androgens). Therefore, in some embodiments, thecombination therapy performs similarly to the individual components whenused alone, but remains effective in presence of rising hormone levels.Accordingly, the combination is improved over the individual componentsalone. Although the cancer killing effect of the combination is similarto the individual components, the duration of efficacy of the treatmentis longer because the cancer does not become resistant to the treatment.This allows the combination therapies to be administered in combinationwith or as an alternative to hormone therapy, a first line therapy, or asecond line or subsequent therapy, and without the need of the cancer tofirst become resistant to the antiandrogen or androgen antagonist (e.g.,abiraterone).

A treatment regimen of the combination therapy can include one ormultiple administrations of antiandrogen or androgen antagonist. Atreatment regimen of the combination therapy can include one or multipleadministrations of Plk inhibitor. In certain embodiments, anantiandrogen or androgen antagonist can be administered simultaneouslywith a Plk inhibitor. Where an antiandrogen or androgen antagonist and aPlk inhibitor are administered at the same time, the antiandrogen orandrogen antagonist and the Plk inhibitor can be in the samepharmaceutical composition.

In some embodiments an antiandrogen or androgen antagonist and a Plkinhibitor are administered sequentially, for example, in two or moredifferent pharmaceutical compositions. In certain embodiments, theantiandrogen or androgen antagonist is administered prior to the firstadministration of the Plk inhibitor. In other embodiments, the Plkinhibitor is administered prior to the first administration of theantiandrogen or androgen antagonist. For example, the antiandrogen orandrogen antagonist and the Plk inhibitor can be administered to asubject on the same day. Alternatively, the antiandrogen or androgenantagonist and the Plk inhibitor are administered to the subject ondifferent days.

The Plk inhibitor can be administered at least 1, 2, 3, 5, 10, 15, 20,24 or 30 hours or days prior to or after administering of theantiandrogen or androgen antagonist. Alternatively, the Plk inhibitorcan be administered at least 1, 2, 3, 5, 10, 15, 20, 24 or 30 hours ordays prior to or after administering of the Plk inhibitor. In certainembodiments, additive or more than additive effects of theadministration of antiandrogen or androgen antagonist in combinationwith one or more Plk inhibitors is evident after one day, two days,three days, four days, five days, six days, one week, or more than oneweek following administration.

Dosage regimens or cycles of the agents can be completely or partiallyoverlapping, or can be sequential. For example, in some embodiments, allsuch administration(s) of the antiandrogen or androgen antagonist occurbefore or after administration of the Plk inhibitor. Alternatively,administration of one or more doses of the antiandrogen or androgenantagonist can be temporally staggered with the administration of Plkinhibitor to form a uniform or non-uniform course of treatment wherebyone or more doses of antiandrogen or androgen antagonist areadministered, followed by one or more doses of Plk inhibitor, followedby one or more doses of antiandrogen or androgen antagonist; or one ormore doses of Plk inhibitor are administered, followed by one or moredoses of antiandrogen or androgen antagonist, followed by one or moredoses of Plk inhibitor, etc., all according to whatever schedule isselected or desired by the researcher or clinician administering thetherapy.

An effective amount of each of the agents can be administered as asingle unit dosage (e.g., as dosage unit), or sub-therapeutic doses thatare administered over a finite time interval. Such unit doses may beadministered on a daily basis for a finite time period, such as up to 3days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15days or up to 20 days or up to 25 days, are all specificallycontemplated.

C. Diseases to be Treated

The combination therapies are typically used to treat cancer, preferablya hormone-sensitive or hormone-dependent cancer that has becomehormone-insensitive. Typically, hormone-sensitive cancers are initiallydependent on a hormone for cancer growth. Altering the cancer's hormonesupply through hormone therapy such as radiation therapy, drugs thatalter hormone production, anti-hormones, aromatase inhibitors,Luteinizing hormone-releasing hormone (LH-RH) agonists and antagonists,or surgery to remove hormone producing tissue or organs, can make thetumors shrink and even lead to cancer remission. However, the effects ofhormone therapy can be limited. Hormone sensitive cancers often becomehormone-insensitive, meaning the cancer is no longer responsive tohormone therapy, although in most cases the tumor is still driven byintracellular hormonal signaling. The combination therapies areparticularly effective for treating hormone-sensitive cancers that havebecome hormone-insensitive. Exemplary cancers include, but are notlimited to, prostate cancer, breast cancer, ovarian cancer, andendometrial cancer.

When hormone therapy is no longer effective, subjects withhormone-insensitive cancers are typically administered a first linetherapy. However, in many cases, the cancer cells also developresistance to the first line therapy. For some hormone-insensitivecancers there are second line therapies available, however, over timethe cancer cells can also develop resistance to the second line therapy.As discussed in more detail below, the combination therapies canre-sensitize cancer cells to a first or second-line therapy. Therefore,in some embodiments, the combination therapies are used to treatsubjects with a hormone-insensitive cancer that is also resistant to afirst line therapy, a second line therapy, or a combination thereof. Insome cases the first line therapy, second line therapy, or a combinationthereof includes the administration of one of the active agents of thecombination therapy without co-administration of the other active agent.Therefore, in some embodiments administration of the combination therapyre-sensitizes the cancer cells to an active agent that was previouslyadministered to the subject as a first or a second line therapy. Inpreferred embodiments, the re-sensitization is effective even in thepresence of rising hormone levels.

In preferred embodiments, the cancer cells can express the androgenreceptor, and/or the cancer is an androgen-sensitive cancer that hasbecome androgen-insensitive (also referred to as an androgen-independentcancer). Androgen-independent cancer is typically a cancer that hasreacquired an ability to grow following temporary suppression of thecancer's ability to grow by inhibiting androgen production or function.In some embodiments, suppression of the cancer's ability to grow refersto suppression of tumor growth or another symptom of the cancer, forexample, amelioration of ostealgia.

Suppression of the cancer's ability to grow using a hormone therapy orphysical castration (e.g., orchiectomy), can be measured using abiochemical assay, for example, measuring a decline in the prostatespecific antigen (PSA) concentration in the blood, or by a morphometricanalysis, for example by computerized tomography (CT), magneticresonance imaging (MRI) or ultrasound. A decline in the blood PSAconcentration effective to reduce an androgen sensitive cancer's abilityto grow is typically a blood PSA concentration of about or below 5ng/mL, about or below 1 ng/ml, about or below 0.5 ng/ml, about or below0.2 ng/ml, about or below 0.1 ng/ml, or undetectable.

A cancer that has reacquired an ability to grow can be an increase intumor growth, the emergence, reemergence, or aggravation of othersymptoms such as ostealgia, new sites of metastasis, or a rise in bloodPSA. A sustained rise in blood PSA concentration observed in the courseof periodic tests can indicate that the cancer has reacquired theability to grow. A blood PSA concentration of, for example, about 5ng/mL or more can also indicate that the cancer has reacquired theability to grow. Because various factors (such as sexual activity) cancause PSA levels to fluctuate, one abnormal PSA test does notnecessarily indicate a problem.

In other embodiments, the cancer does not express or over express AR orER.

1. Prostate Cancer

The cancer can be prostate cancer. Prostate cancer is the mostfrequently diagnosed malignancy in men in Western countries. Whilelocalized prostate cancer can be effectively treated with surgery orradiation therapy, metastatic PCa still remains incurable. For locallyadvanced or widespread disease, suppressing the tumor growth by hormoneablation therapy represents the common first therapeutic option(Beltran, et al., European Urology, 60:279-290 (2011)). Although initialtherapy can lead to long-term remission, development of hormone ablationresistance can eventually occur, a standing referred to ascastration-resistant prostate cancer (CRPC). Therefore, in someembodiments, the subject has a CRPC. Unlike early prostate cancer, CRPCis an aggressive disease that progresses despite castrate levels oftestosterone (≦50 ng/ml). It is diagnosed by one or more of thefollowing discussed above for androgen-insensitive cancers (e.g.,sustained rise in serum levels of prostate-specific antigen (PSA),progression of pre-existing disease, the appearance of new metastases,or a combination thereof).

Subjects with CRPC are typically administered a first line therapy.Docetaxel and sipuleucel-T are exemplary first line treatment optionsfor patients with CRPC. Second-line treatments following first linetreatment failure include cabazitaxel and abiraterone acetate. Thehistory of first and second line therapeutic options for subjects withCRPC are reviewed in Shapiro and Tareen, Expert Rev. Anticancer Ther.,12(7):951-964 (2012) and Heidegger, et al., J. Steroid Biochem. Mol.Biol., 138(100): 248-256 (2013), which are both specificallyincorporated by reference herein in their entireties. The combinationtherapies can be administered to subjects which have previously beenadministered a first line therapy for CRPC and/or a second line therapyfor CRPC. In a particular embodiment, the combination therapy isadministered to subject when a first line therapy and/or a second linetherapy have become ineffective to treat or prevent progression of thecancer.

In a particular embodiment, the combination therapy is administered to asubject that was previously administered an agent that targets andinhibits androgen receptor activity. The agent can target androgenreceptor activity directly, or indirectly, for example by inhibitingandrogen synthesis. In a particular preferred embodiment, the subjectwas previously administered an abiraterone-based therapy such as ZYTIGA®(abiraterone acetate). Abiraterone has been administered to subjects asa first line therapy and as a second line therapy, typically followingchemotherapy, for treatment of CRPC. The data presented in the workingExamples below illustrates that the combination therapies are effectiveto treat cancers that have become resistant to abiraterone.

2. Breast Cancer

The cancer can be breast cancer, preferably a breast cancercharacterized by breast cancer cells that expresses the androgenreceptor. Androgens play a role in normal breast physiology and androgenreceptor (AR) signaling is recognized as an important contributor inbreast carcinogenesis (Garay, et al., Am. J. Cancer Res., 2(4):434-445(2012)). The androgen receptor is expressed in most breast cancers, andalthough the mechanism underlying the androgen receptor's role in cancerprogression remains unclear, it has been identified as a potentialtherapeutic target for breast cancer treatment. Therefore, in someembodiments, the combination therapy is administered to treat a breastcancer, preferably, but not limited to, an androgen receptor-positivebreast cancer.

The success of estrogen receptor/progesterone receptor (ER/PR) and HER2targeted therapies has shifted interest in androgen receptor to thosebreast cancers that lack ER/PR and/or HER2 expression, often referred toas “triple negative breast cancer” or “triple negative disease”. Inaddition, AR targeted therapies may also be important for breast cancersthat have developed resistance to current hormone and HER2 directedtherapies. Therefore, in some embodiments, the breast cancer lacks ER,PR, or HER2 expression, or a combination thereof (e.g., triple negativedisease), is a hormone-insensitive cancer, is resistant to a HER2directed therapy, or any combination thereof.

It has also been shown both in vitro and in vivo that combinatorialtherapy targeting both the MAP kinase pathway and AR is an effectivemeans of reducing tumor cell viability and tumor burden (Naderi and Liu,Cancer Lett., 298: 74-87 (2010)). Therefore, in some embodiments, thecombination therapies include an inhibitor of the MAP kinase pathway.

3. Other Cancers

The compositions and methods described can be used to treat multipletypes of cancer. It has been established that an androgen receptorantagonist or anti-androgen, or a derivative, analog or prodrug, or apharmacologically active salt thereof in combination with one or moreinhibitors of Plk can give rise to profound greater than additivekilling of cancers that are not associated with consistent expression ofAndrogen receptor signaling or other steroid hormone or growth factors.Therefore, multiple non-hormonal cancers can be treated using thecompositions and methods described herein.

The combination is particularly effective in treating cancerscharacterized by up-regulated expression of genes that are involved inthe Retinoic Acid Receptor (RA) signaling pathway, specifically genesincluding the Retinoic Acid Receptor Alpha (RARA); Retinoic AcidReceptor Gamma (RARG); Retinol Dehydrogenase (ADH4); and RetinaldehydeReductase (DHRS3). Cancers that have been identified as havingup-regulated expression of genes that are involved in the Retinoic AcidReceptor (RA) signaling pathway include multiple types of cancers,including but not limited to prostate, breast, and pancreatic cancers.

Pancreatic cancers include pancreatic adenocarcinoma or pancreaticexocrine cancer, and often have a poor prognosis, even when diagnosedearly. Pancreatic cancer typically spreads rapidly and is seldomdetected in its early stages, and pancreatic carcinoma is a leadingcause of cancer death. Signs and symptoms can include upper abdominalpain; bowel obstruction; back pain; yellow discoloration of the skin andwhites of the eye (i.e., jaundice); reduced appetite; weight loss;depression; and blood clots. Symptoms may not appear until pancreaticcancer is quite advanced and complete surgical removal is not possible.

Gene expression profiles for cancer cells within a tumor or for cellswithin the tumor microenvironment can be determined in vitro or in vivoby any means known in the art. Genomic databases can also be used as aguide for the selection of pharmacologic vulnerabilities to genomicpatterns. Such databases include the Cancer Cell Line Encyclopedia(CCLE), including gene expression profile information for human cancercell lines (Stransky, et al. Nature 483, 603-307 (2012)).

A representative, but non-limiting, list of cancers that the disclosedcompositions and methods can be used to treat include lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloidleukemia, bladder cancer, brain cancer, nervous system cancer, head andneck cancer, squamous cell carcinoma of head and neck, kidney cancer,lung cancers such as small cell lung cancer and non-small cell lungcancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,skin cancer, liver cancer, melanoma, squamous cell carcinomas of themouth, throat, larynx, and lung, colon cancer, cervical cancer, cervicalcarcinoma, epithelial cancer, renal cancer, genitourinary cancer,pulmonary cancer, esophageal carcinoma, head and neck carcinoma, largebowel cancer, hematopoietic cancers; testicular cancer; colon and rectalcancers, and pancreatic cancer.

D. Compositions for Use in Disease Treatment

Compositions for use in the treatment of the disclosed diseases are alsoprovided. For example, a composition including an antiandrogen orandrogen antagonist for use in a method of treating a subject withcancer, wherein the subject is one whom a composition including a Plkinhibitor has previously been or is concurrently being administered andwherein the response achieved following the administration ofantiandrogen or androgen antagonist is greater than the responseachieved by administering either the antiandrogen or androgen antagonistalone or the Plk inhibitor alone are disclosed.

In another embodiment, a composition including a Plk inhibitor for usein a method of treating a subject with cancer, wherein the subject isone whom a composition including an antiandrogen or androgen antagonisthas previously been or is currently being administered and wherein theresponse achieved following the administration of the Plk inhibitor isgreater than the response achieved by administering either theantiandrogen or androgen antagonist alone or the Plk inhibitor alone isprovide. Suitable compositions, cancers to be treated, dosage regimes,and responses achieved by administering the combinations are discussedat length above. In particular embodiments the subject may have beenpreviously administered one or more of the drugs, but not incombination.

Furthermore, it will be appreciated as discussed above, that the cancermay have developed a resistance to the previously administered activeagent the active agent is administered in the absence of thecombination. Therefore, in some embodiments, the subject populationbeing treatment is defined as one in which the cancer being treated isresistant or insensitive to one or the other of the active agent whenadministered alone.

IV. Kits

Medical kits are also disclosed. The medical kits can include, forexample, a dosage supply of an antiandrogen or androgen antagonist, apolo-like kinase inhibitor, or a combination thereof in separately ortogether in the same admixture. The active agents can be supplied alone(e.g., lyophilized), or in a pharmaceutical composition. The activeagents can be in a unit dosage, or in a stock that should be dilutedprior to administration. In some embodiments, the kit includes a supplyof pharmaceutically acceptable carrier. The kit can also include devicesfor administration of the active agents or compositions, for example,syringes. The kits can include printed instructions for administeringthe compound in a use as described above.

EXAMPLES Example 1 Prostate Cancer Cells are Sensitive to Plk1Inhibition

Materials and Methods

Prostate cancer cell lines (LNCaP and C4-2) were seeded into 96-wellplates in RPMI-1640 medium containing 10 fetal-bovine serum depleted ofandrogen by activated charcoal-dextran (csFBS). Twenty four hours later,the indicated concentration of BI2536 and R1881 were added, representingtime zero. Viability of the prostate cancer cells was assessed usingCell-Titer Glo™ according to the manufacturer's instructions.

Results

An experiment was designed to test the effect of Plk1 on prostate cancercells. The androgen-sensitive cells (LNCaP) and castrate resistantprostate cancer cells (CRPC) (C4-2) were treated with increasingconcentrations of BI2536 in the presence and absence of syntheticandrogen (R1881). The results, shown in FIG. 3A shows that viability ofboth cell lines is reduced in the presence of BI2536, but the viabilitycan be somewhat rescued in the presence of synthetic androgen.

FIG. 3B shows the results of an assay measuring the proliferation ofcells over time in the presence of 5 nM BI2536. The results indicatethat proliferation of both cell lines is reduced in the presence ofBI2336, but proliferation can be somewhat rescued in the presence ofsynthetic androgen Thus increased androgen receptor activity can driveproliferation despite the loss of Plk1 activity.

Together, the results indicate that CRPC cells are particularlydependent on Plk1 activity when the AR is not sufficiently stimulated.

Example 2 The Effect of Co-Treatment with BI2536 and Abiraterone onProstate Cancer Cells is More than Additive

Materials and Methods

For FIG. 4, CRPC cells (C4-2) were seeded into 96-well plates inRPMI-1640 medium containing 10% FBS (not stripped withcharcoal-dextran). 24 hours later the indicated concentrations ofAbiraterone and/or BI2536 were added, representing day 0. Viability wasmeasured for the subsequent five days using Cell-Titer Glo™ according tothe manufacturer's instructions.

Dose response curves presented in FIG. 5A-C were measured by seedinginto 96 well plates CRPC cells (C4-2 and 22RV1) and androgen dependentprostate cancer cells (LNCaP) in RPMI 1640 containing 10% csFBS or FBS,respectively. Twenty four hours later the indicated concentrations ofAbiraterone and 5 nM BI2536 were added. Cell viability was assessedafter 5 days using Cell-Titer Glo™ according to the manufacturer'sinstructions.

Results

Experiments were designed to test the effect of co-administration ofBI2536 and abiraterone on the proliferation prostate cancer cells. CRPCcells (C4-2) were treated with vehicle (DMSO), 5 nM BI2536, 10 μMabiraterone, or 5 nM BI2536 in combination with 10 μM abiraterone andproliferation was monitored over 5 days post-administration. Theseexperiments were done in medium containing FBS and physiological levelsof androgen. The results, shown in FIG. 4 shows that relative levels ofproliferation are reduced over time in the presence of 5 nM BI2536,alone, and 10 μM abiraterone alone, but synergistically reduced when thetwo agents are administered in combination. Moreover, the combination ofabiraterone and BI2536 resulted in a decrease in cell viability overtime, indicating a loss of cell number as opposed to a cytostaticeffect.

FIGS. 5A-5C show the results of assays measuring the % viability ofcells (LNCaP—androgen dependent; 22RV1—castrate resistant; andC4-2—castrate resistant) after five days in the presence (

) or absence (

) of 10 nM BI2536 and increasing concentrations of abiraterone. Theexpected viability if abiraterone and BI2536 were acting in an additivemanner is also indicated (

). The results indicate that, particularly with CRPC cells, thereduction in % viability of cells treated with both agents is reduced toa greater degree than predicted based an additive effect of thecombination.

Together, the results indicate that the effect of the combination ofBI2536 and abiraterone on prostate cancer cells, including castrateresistant prostate cancer cells is more than additive. While the CRPCcells are relatively resistant to Abiraterone in the absence of BI2536,their sensitivity is essentially equivalent to androgen dependent cellswhen a low concentration of Plk inhibitor is added. These results canlead to a conclusion that castrate resistant prostate cancer cells aresensitized to abiraterone by BI2536.

Example 3 The Effect of Co-Treatment with BI2536 and Abiraterone onBreast Cancer Cells is More than Additive

Materials and Methods

The breast cancer cell lines AU565 (expresses the AR) and BT20 (does notexpress the AR) were seeded into 96-well plates in DMEM mediumcontaining 10% FBS. Twenty four hours later the indicated concentrationsof Abiraterone and 5 nM BI2536 were added. Cell viability was assessedafter 5 days using Cell-Titer Glo™ according to the manufacturer'sinstructions.

Results

Experiments were designed to test the effect of co-administration ofBI2536 and abiraterone on breast cancer cells. FIGS. 6A and 6B show theresults of assays measuring the % viability of the AU565 and BT20 cellsin the presence (

) or absence (

) of BI2536 and increasing concentrations of abiraterone. The expectedoutcome if these agents were acting in an additive manner is alsoindicated (

). The results indicate % viability of cells treated with both agents isreduced to a greater degree than predicted based an additive effect ofthe combination. Together, the results indicated that the effect of thecombination of BI2536 and abiraterone on breast cancer cells is morethan additive. These results can lead to a conclusion that some breastcancer cells are sensitized to abiraterone by BI2536. Moreover, becausethe nature of prostate cancer and breast cancer is substantiallydifferent, the observation that BI2536 synergizes with abiraterone inboth cell types indicates that this is a fundamental characteristic ofthese signaling proteins and not a peculiarity of the relatively fewCRPC cell lines available.

Example 4 The Effect of Co-Treatment with BI2536 and Abiraterone is nota Result of G2/M Arrest

Materials and Methods

CRPC cells (C4-2 and 22RV1) and androgen dependent prostate cancer cells(LNCaP) were seeded in 96-well plates using RPMI 1640 containing 10%csFBS or FBS, respectively. Twenty four hours later the indicatedconcentrations of Abiraterone and 5 nM Docetaxel were added. Cellviability was assessed after 4 days using Cell-Titer Glo™ according tothe manufacturer's instructions.

Results

Experiments were designed to investigate the mechanism underlying theeffect of co-administration of BI2536 and abiraterone. Previouslyreported experiments have indicated that BI2536 can induce cell cyclearrest.

FIGS. 7A-7C show the results of assays measuring the % viability ofcells (LNCaP—androgen dependent; 22RV1—castrate resistant; andC4-2—castrate resistant) over time in presence of 5 nM docetaxel (

) or absence (

) of 5 nM docetaxel and increasing concentrations of abiraterone.Docetaxel is a taxane, used in the treatment of CRPC, which prevents thedepolymerization of microtubules resulting in mitotic arrest andsubsequent death. This is thought to be the basis for its ability totarget highly proliferative cancer cells. Expected results if the drugswere acting in an additive manner are also indicated (

). Docetaxel, and the mitotic arrest caused by treatment with docetaxel,did not synergize with Abiraterone in prostate cancer cells, as judgedby the overlapping observed and expected data.

This result can lead to a conclusion that it is not simply a G2/M arrestthat leaves cells treated with BI2536 more sensitive to abiraterone. Theresults can also lead to a conclusion that the effect is not due to aloss of microtubule dynamics, which also occurs during loss of Plk1kinase activity.

Example 5 Plk1 is not a Generic Upstream Activator of the AR

Methods

Experiments were designed to investigate the molecular mechanism for theeffects of combination Abiraterone/Plk1 inhibitor Treatment. C4-2 CRPCcells were grown in FBS and subjected to Abiraterone alone, or BI2536alone, or the combination of Abiraterone and BI2536 for 24 hours. RNAwas isolated for analysis by qPCR.

Results

Abiraterone substantially decreased the amount of PSA expression,however, the Plk1 inhibitor BI2536 did not suppress PSA expression aloneand the combination of Abiraterone and BI2536 did not reduce PSAexpression to any greater extent than Abiraterone alone (see FIG. 8).

These data indicated that Plk1 is not a generic activator of AR-drivengene expression, since the presence or absence of a potent Plk1inhibitor has no effect on AR-driven PSA gene expression.

Instead, the data suggested there must be some other mechanism for thegreater than additive effect of Plk1 inhibitor/Abiraterone than simplyaltering the AR transcriptional activity in a global manner. Thus,although Plk1 is not a generic regulator of the AR, perhaps Plk1 insteadcooperates with the AR to regulate a subset of AR-dependent genes.

Example 6 Molecular Mechanism for Combination Abiraterone/Plk1 InhibitorTreatment Involves Transcription Factors Elk1 and Nrf1/2

Methods

The molecular bases for combination Abiraterone/Plk1 inhibitor Treatmentwere examined using RNA-Seq experiments.

Since Plk1 inhibition results in mitotic arrest, but other clinicallyused drugs that cause mitotic arrest, such as taxanes, do not synergizewith Abiraterone for tumor killing, the design of RNA-Seq-based geneexpression experiments was carefully tailored to avoid being mis-lead bygene expression changes from the mitotic arrest phenotype. To eliminatethe convoluted effects mitotic arrest exerts on transcription, which arenot solely causative, doses of Plk1 inhibitor and Taxanes that resultedin comparable mitotic arrest were determined experimentally under sixconditions (DMSO, 10 μM Abiraterone, BI2536 (Plk1 inhibitor),Abiraterone+BI2536, Docetaxol (a mitotic arrest control), andAbiraterone+Docetaxol (as a mitotically-arrested non synergistic controlwith Abiraterone), see FIG. 9A).

The strongly AR-dependent gene, PSA, was used as a means to determinethe optimal time point for RNA isolation post drug treatment. Based onthese analyses using changes in PSA expression as a function of time, 16hours after treatment was selected as the earliest time point whereAbiraterone-induced suppression of AR-driven gene expression could bedetected (FIG. 9B).

RNA-Seq analysis of C4-2 cells was carried out to identify gene whoseexpression is changed specifically in cells treated with bothAbiraterone and a Plk1 inhibitor. C4-2 cells were grown in mediacontaining FBS and treated with the indicated drugs: 10 μM Abiraterone(Abi), 2.5 nM BI2536 (BI) and/or 1 nM Docetaxel (DTX) for 16 hours intriplicate prior to RNA-isolation. Reads (400 million) were aligned tothe human genome and gene level expression data generated. The data wasthen transformed into robust z-scores. Differentially expressed genes(DEGs) of interest were identified using the statistical analysis ofmicroarrays (SAM) package based on the following criteria: Significantdifference between Abi vrs Abi BI; no significant difference between Abivrs Abi DTX; and no significant difference between DMSO and Abi.

Computational analysis was performed for both up-regulated anddown-regulated gene sets using both the DAVID (Database for Annotation,Visualization and Integrated Discovery) and GSEA (Gene Set EnrichmentAnalysis) algorithms to identify potential transcription factors whoseactivities were uniquely modulated by the combined Plk1inhibition/Abiraterone treatment.

Results

The experiments identified Genes whose expression changed (induced orrepressed) at the 16 hour time point specifically in response to by thecombination of Abiraterone and Plk1 inhibition by BI2536, but not inresponse to Abiraterone alone.

The data identified multiple genes of interest. Within this list ofgenes there is a highly significant enrichment of genes which have beenfound to contain binding sites for the transcription factors Elk1 andNrf1/2 adjacent to their transcriptional start site. Thus, the combinedanalyses implicated both Elk1 and Nrf1/2 as critical targets forPlk1i/Abiraterone effects. Nrf1/2 is a particularly interestingpotential mechanistic target that rationalizes the greater than additivecombinatorial effect.

Example 7 A Significant Subset of Breast Cancer Cell Lines DisplaySynergistic Killing by Combination Abiraterone Plus Plk1 Inhibition

Methods

Experiments were conducted to quantify the greater than additive effectbetween Abiraterone and Plk1 inhibitors.

A very comprehensive series of experiments was performed in 23 celllines using combinations of 9 different doses of the Plk1 inhibitor and6 different doses of Abiraterone. Cell lines were subjected to a doseresponse matrix of increasing concentrations of Abiraterone (0 to 10 μM)and BI2536 (0 to 15 nM) and assessed for viability after 3 days usingCell Titer Glo™. Greater than additive killing by the combination ofPlk1 inhibitors plus abiraterone was measured after 3 days andquantified by summing the difference between the areas under the curve(AUC) for expected killing assuming only drug additivity (

; FIG. 10A-10I) and the measured cell killing (

; FIG. 10A-10I), for increasing concentrations of Abiraterone for eachdose of BI2536 (Total dAUC) (see FIGS. 10A-10I).

These numbers were then correlated with mRNA expression datasetsobtained from the Cancer Cell Line Encyclopedia (CCLE) and the entirematrix was transformed into robust scores.

Results

These experiments revealed that a substantial subset of the breastcancer cell lines tested showed greater than additive killing. The dataalso provided information on the sensitivity of each cell line toabiraterone alone. Further more than additive effects for some celllines is apparent only after 5 days of treatment.

The relative expression of the estrogen receptor alpha (ERα), the AR,the progesterone receptor (PGR) and Her2 were plotted according to theCancer Cell Line Encyclopedia (CCLE) (FIG. 11). The relative position ofgenes on the plot demonstrated that a significant portion of breastcancer cell lines express the androgen receptor. However, the breastcancer cell lines that displayed greater than additive effect ofAbiraterone and Plk1 inhibition (indicted by full arrows in FIG. 11) donot appear to depend on AR signaling or show any other consistentexpression of these steroid hormone or growth factor genes. In fact, themajority of the synergistic cell lines are derived from triple-negativebreast tumors (TNBCs), and the sensitivity of the cells to combinationtreatment does not appear to correlate with androgen receptorexpression, or with estrogen receptor expression (FIG. 11). Furthermore,profound greater than additive effect was also observed in somenon-hormonal tumor types including pancreatic cancer. This haspotentially important clinical implications since effective chemotherapyfor TNBCs, and pancreatic cancer remains a high-priority unmet need.These data clearly indicated that Abiraterone, in combination with Plk1inhibition, can have utility far beyond prostate cancer.

Example 8 Tumor Cell Lines that are Sensitive to CombinedAbiraterone/Plk1 Inhibition Treatment have a Gene Expression Signature

Methods

Experiments were conducted to determine the molecular signature of up-and down-regulated genes whose expression correlates very well withsensitivity to combined Abiraterone/Plk1 inhibition treatment. Tounderstand why certain cancer cell lines showed marked greater thanadditive effects to combination Abiraterone/Plk1 inhibition treatment,cell lines were ranked by “synergy score” (i.e., observed additiveeffect of combination Abiraterone/Plk1 inhibition treatment), andcross-correlated with gene expression data.

Comprehensive gene expression values for each of the cell lines wereobtained from the Cancer Cell Line Encyclopedia (CCLE), and used forclustering analysis based on synergy score ranking. Correlation betweenthe synergy score ranking and expression of any given gene wascalculated by Pearson correlation coefficients and the genes were thenplotted on a graph and ranked in order from most to least correlatedwith the observed amount of synergy score. These data were then used todetermine which genes were associated with synergy.

Results

Gene set enrichment analysis performed on the sensitivity-ranked celllines indicated that synergistic cell lines showed a less mesenchymalpattern of gene expression, up-regulation of cholesterol biosynthesis,and decreased expression of mitochondrial/oxidative phosphorylationgenes.

Importantly, within the top 30 genes whose up-regulation correlated withstrong synergy score to combination treatment are 4 components of theRetinoic Acid Receptor (RA) signaling pathway. Cell lines whichdisplayed the most greater than additive effect had high expressionlevels of several genes involved in retinoic acid signaling, includingRetinoic Acid Receptor Alpha (RARA); Retinoic Acid Receptor Gamma(RARG); Retinol Dehydrogenase (ADH4); and Retinaldehyde Reductase(DHRS3). Of these four genes, RARA was the gene most highly correlatedwith synergy score, with a Pearson correlation coefficient of 0.806,p=0.000031.

The RA signaling pathway is a nuclear hormone signaling pathway similarto, but distinct from the AR and ER pathways, that has pleiotropic rolesin cell growth and differentiation. Abnormalities in RA receptorsignaling have been clearly implicated in breast and hematologicalmalignancies. The pathway appears to have differential effects in normalversus cancer cells, and in certain cancer types, RA signaling appearsto be important for proliferation and resistance to apoptosis (Noy, AnnuRev Nutr, pp. 201-217 (2010)). It has been shown that retinoic acidsignaling markedly potentiated the ability of Plk1 inhibitors to killcells. (Liu-Sullivan, et al., Oncotarget, pp. 1254-1264 (2011)).Furthermore, there is clear data that RA signaling suppressesNrf1/2-driven gene expression, and that signaling through the androgenreceptor suppresses the RA signaling that regulates Nrf1/2-driven genes(Schultz, et al., PLoS ONE, pp. e87204 (2014); Wang, et al., PNAS USA,pp. 19589-19594 (2007)).

Thus, these data strongly suggest a model for Abiraterone action inwhich Abiraterone inhibition of androgen signaling results inup-regulation of RA signaling due to nuclear hormone pathway cross-talk.The RA up-regulation makes the cells sensitive to Plk1 inhibition.

In addition, the combined effects of Plk1 inhibition and RAup-regulation specifically modulates Nrf1/2-driven genes, many of whichare involved in mitochondrial oxidative phosphorylation and anti-oxidantresponses, thereby explaining the gene expression data for combinationtreatments, as well as the sensitivity of specific tumor cell types.This model can provide a means to select tumors for combinatorialtreatment, provide a direct measurement of treatment response that couldbe followed clinically, rationalize the mechanism for Abirateroneresistance, and provide a new series of drug targets (the RA pathway)that could be used to enhance Abiraterone's effectiveness as anantitumor agent.

Example 9 The p38MAPK-MK Pathway Influences the Response of Tumors toAbiraterone

Methods

Experiments were conducted to investigate the role of the p38MAPK-MK2pathway in prostate cancer. Western blotting was used to investigateup-regulation of MK3 and MK5 protein levels in LNCaP cells after MK2knock-down using shRNA. No change was observed in 22Rv1 or C4-2 cells.RiLNCaP cell viability was measured using Cell-Titer Glo in response to10, 20 or 40 μM of the p38MAPK inhibitor SB203580 in the presence orabsence of 10 μM Abiraterone (FIG. 12).

Experiments were also conducted to determine the effects of p38inhibition on Abiraterone sensitivity in Prostate Cancer cell lines.Cell viability of PCa cell lines to increasing doses (10, 20 and 40 μM)of the p38MAPK inhibitor SB203580, as measured using Cell Titer Glo(FIG. 13A). The effect of increasing doses of SB203580 (μM) on theextent of PCa cell killing by 10 μM Abiraterone was also assessed (FIG.13B). The effects of the two drugs appear to be additive.

The data indicated that MK2 inhibition, which partially suppresseshsp27phosphorylation, had little effect on survival of prostate cancercell lines. These results were unusual, since there is a reasonablysolid body of evidence indicating that phospho-hsp27 is required fornuclear chaperoning of the activated form of the androgen receptor. Thelack of effect of MK2 disruption on PCa survival was attributed topossible up-regulation of MK3 and/or MK5 kinases, both of which mightpartially subsume the role of MK2 in hsp27 phosphorylation.

Up-regulation of both MK3 and MK5 was observed in LNCaP cells but not in22Rv1 or C4-2 cells. Importantly, inhibition of the upstream kinase,p38MAPK, by small molecule inhibitors resulted in cell death in the MK2knock-down LNCaP cells, in agreement with the hypothesis that thepathway was re-wired.

Furthermore, p38MAPK inhibition alone was able to cause small amounts ofcell death in all of the cell lines, but more marked levels of deathwere only observed at rather high levels of the p38 inhibitor (FIG.13A). The p38MAPK inhibitor also modestly enhanced the ability ofAbiraterone to kill all of the PCa cell lines (FIG. 13B).

Further experiments were conducted to include co-culture usingendothelial cells. These data indicated that conditioned media fromHUVEC cells partially protected the PCa cells from Abiraterone ortaxane-induced cell death. The data further indicated that MK2 signalingin the endothelial cells is necessary for the Abiraterone andtaxane-resistance.

Thus, the p38MAPK-MK2 pathway appears to play complex roles inintegrating the tumor response to Abiraterone through effects in boththe tumor cells and the tumor microenvironment. In addition, there isevidence that the p38MAPK/MK2 pathway can also influences the RAsignaling pathway (Oianni, et al., The EMBO journal 25, 739-751 (2002);Gianni, et al., The EMBO journal 21, 3760-3769 (2002)).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method of treating a patient with cancer comprisingadministering to the patient an effective amount of abiraterone orabiraterone acetate in combination with an effective amount of apolo-like kinase inhibitor (PLK), wherein the abiraterone or abirateroneacetate and the polo-like kinase inhibitor are administered simultaneousor wherein the abiraterone or abiraterone acetate is administered to thepatient 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours, 1, 2, 3, 4, 5, 6,or 7 days, 1, 2, 3, or 4 weeks, prior to administration of the polo-likekinase inhibitor to the patient, and wherein administration of thecombination of abiraterone or abiraterone acetate and the polo-likekinase inhibitor is effective to reduce cancer cell proliferation orviability in the patient to a greater degree than administering to thepatient the same amount of abiraterone or abiraterone acetate alone orthe same amount of polo-like kinase inhibitor alone.
 2. The method ofclaim 1, wherein the dosage of abiraterone acetate is 250 mg-1,500 mg.3. The method of claim 1, wherein the class of polo-like kinaseinhibitor is selected from the group consisting of dihydropteridinones,pyridopyrimidines, aminopyrimidines, substituted thiazolidinones,pteridine derivatives, dihydroimidazo[1,5-f]pteridines, metasubstitutedthiazolidinones, benzyl styryl sulfone analogues, stilbene derivatives,and combinations thereof.
 4. The method of claim 3, wherein thepolo-like kinase inhibitor is selected from the group consisting ofBI2536, Volasertib (BI 6727), GSK461364, HMN-176, HMN-214, rigosertib(ON-01910), MLN0905, TKM-080301, TAK-960, NMS-1286937 or Ro3280.
 5. Themethod of claim 4, wherein the polo-like kinase inhibitor inhibitor isBI2536.
 6. The method of claim 5, wherein the dosage of BI25236 isbetween one and 500 mg.
 7. The method of claim 1, wherein the cancercells are selected from the group consisting of breast cancer cells,prostate cancer cells and pancreatic cancer cells.
 8. The method ofclaim 1, wherein the reduction in cancer cell proliferation or viabilityin the subject with cancer is more than the additive reduction achievedby administering the abiraterone or abiraterone acetate alone or thepolo-like kinase inhibitor alone.
 9. The method of claim 1, wherein thecancer cells are insensitive to abiraterone when abiraterone acetate isadministered without co-administration of the polo-like kinaseinhibitor.
 10. The method of claim 1, wherein the abiraterone orabiraterone acetate is administered to the subject 1, 2, 3, 4, 5, 6, 8,10, 12, 18, or 24 hours, prior to administration of the polo-like kinaseinhibitor to the subject.
 11. The method of claim 1, further comprisingadministering to the subject one or more additional active agentsselected from the group consisting of a steroid, a chemotherapeuticagent, an anti-infective agent, and combinations thereof.
 12. The methodof claim 11, wherein one additional active agent is the steroidprednisone.
 13. The method of claim 11, wherein one additional activeagent is the chemotherapeutic agent is docetaxel.
 14. The method ofclaim 1, further comprising surgery or radiation therapy.
 15. The methodof claim 1, wherein one or more genes involved in retinoic acidsignaling are selected from the group consisting of Retinoic AcidReceptor Alpha (RARA); Retinoic Acid Receptor Gamma (RARG); RetinolDehydrogenase (ADH4); and Retinaldehyde Reductase (DHRS3).
 16. Themethod of claim 1, wherein the cancer is characterized by expression,over-expression or up-regulation of genes driven by the Nrf1/2 or Elk1transcription factors following treatment.
 17. The method of claim 1,further comprising the step of selecting a subject having a cancercharacterized by expression of genes involved in the Retinoic AcidReceptor (RA) signaling pathway.
 18. The method of claim 1, wherein thecancer is selected from the group consisting of prostate cancer, breastcancer and pancreatic cancer.